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Published on: November 6, 2013
Identification and characterization of a multispecific monoclonal antibody G2 against chicken prion protein
Yuji O Kamatari1, Shinri Ohta, Yasuo Inoshima
1Life Science Research Center, Gifu University, 1-1 Yanagido, Gifu, 501-1194, Japan.
Abstract:
We previously generated a monoclonal antibody (mAb), G2, by immunizing mice with Residues 174-247 of the chicken prion protein (ChPrP(C) ). In this study, we found that G2 possessed an extremely unusual characteristic for a mAb; in particular, it could react with at least three proteins other than ChPrP(C) , the original antigenic protein. We immunoscreened a complementary DNA library from chicken brain DNA and found three proteins (SEPT3, ATP6V1C1, and C6H10orf76) that reacts with G2. There were no regions of amino acid sequence similarity between ChPrP(C) and SEPT3, ATP6V1C1, or C6H10orf76. We selected ATP6V1C1 as a representative of the three proteins and identified the epitope within ATP6V1C1 that reacts with G2. The amino acid sequence of the G2 epitope within ATP6V1C1 (Pep8) was not related to the G2 epitope within ChPrP(C) (Pep18mer). However, enzyme-linked immunosorbent assay, surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) experiments indicated that these two peptides have similar binding affinity for G2. The apparent KD values of Pep18mer and Pep8 obtained from SPR experiments were 2.9 × 10(-8) and 1.6 × 10(-8) M, respectively. Antibody inhibition test using each peptide indicated that the binding sites of the two different peptides overlapped each other. We observed that these two peptides substantially differed in several binding characteristics. Based on the SPR experiments, the association and dissociation rate constants of Pep18mer were higher than those of Pep8. A clear difference was also observed in ITC experiments. These differences may be explained by G2 adopting different binding conformations and undergoing different binding pathways.
Insights
A novel monoclonal antibody (mAb), G2, unexpectedly binds to multiple proteins beyond its original target, chicken prion protein (ChPrP(C)). This cross-reactivity, despite lacking sequence similarity, reveals complex antibody-epitope interactions and potential for broader applications.
Area of Science:
- Immunology
- Biochemistry
- Molecular Biology
Background:
- Monoclonal antibodies (mAbs) are typically highly specific reagents.
- The generation of mAb G2 involved immunization with chicken prion protein (ChPrP(C)).
- Unusual cross-reactivity of mAbs can present challenges and opportunities in research.
Purpose of the Study:
- To investigate the unexpected cross-reactivity of mAb G2 with proteins other than ChPrP(C).
- To identify the specific proteins that react with mAb G2.
- To characterize the binding epitopes and interactions between mAb G2 and its targets.
Main Methods:
- Immunoscreening of a chicken brain complementary DNA library.
- Identification of reacting proteins using molecular biology techniques.
- Epitope mapping and characterization using enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC).
Main Results:
- mAb G2 reacted with three novel proteins: SEPT3, ATP6V1C1, and C6H10orf76, despite no sequence homology with ChPrP(C).
- Distinct epitopes on ChPrP(C) (Pep18mer) and ATP6V1C1 (Pep8) bound to mAb G2 with similar affinities (apparent KD values of 2.9 × 10⁻⁸ M and 1.6 × 10⁻⁸ M, respectively).
- Kinetic analysis (SPR) and thermodynamic analysis (ITC) revealed differences in binding rates and conformations, suggesting distinct binding pathways.
Conclusions:
- mAb G2 exhibits significant cross-reactivity, binding to unrelated proteins through distinct but conformationally similar epitopes.
- The study highlights the potential for antibodies to recognize structurally analogous epitopes on proteins lacking sequence similarity.
- These findings expand our understanding of antibody-antigen interactions and suggest potential for G2 in broader biological applications.
