Molecular characterization of chicken prion proteins by C-terminal-specific monoclonal antibodies

Ishiguro, Naotaka; Inoshima, Yasuo; Sassa, Yukiko; Takahashi, Toyomi

doi:10.1016/j.vetimm.2008.11.025

Cited by 9 publications

(20 citation statements)

References 12 publications

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“…Detailed investigations about the localization of ChPrP C in chicken neural cells have been limited because of the lack of specific antibodies directed against ChPrP C epitopes . Therefore, recombinant ChPrP (rChPrP) was produced in bacteria, and several mouse monoclonal antibodies (mAbs) against rChPrP were isolated …”

Section: Introductionmentioning

confidence: 99%

Identification and characterization of a multispecific monoclonal antibody G2 against chicken prion protein

et al. 2014

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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 K D values of Pep18mer and Pep8 obtained from SPR experiments were 2.9 3 10 28 and 1.6 3 10 28 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.

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Section: Introductionmentioning

confidence: 99%

Identification and characterization of a multispecific monoclonal antibody G2 against chicken prion protein

et al. 2014

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“…After washing in PBS, they were incubated with 2% normal horse serum, and then incubated in PBS containing a mouse anti-prion protein antibody (13, 1:200), 0.3% Triton X-100, and 1% normal horse serum at 4 8C for 2 days. The specificity of the antibody has been reported elsewhere and the antibody recognize amino acid sequence of Arg 161 -Ser 164 (Ishiguro et al, 2009). The sections were rinsed with PBS, incubated with a biotinylated horse anti-mouse IgG (Vector Laboratories Inc., Burlingame, CA, USA; 1:500) for 1 h at room temperature, and finally incubated in avidin-biotin-horseradish perioxidase complex (ABC Elite Kit; Vector Laboratories Inc.) for 1 h at room temperature.…”

Section: Immunohistochemistrymentioning

confidence: 94%

Distribution of the cellular prion protein in the central nervous system of the chicken

Atoji

Ishiguro

2009

Journal of Chemical Neuroanatomy

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“…Protein samples were subjected to 12% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, and separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane for reaction with G2 …”

Section: Methodsmentioning

confidence: 99%

“…Synthesized peptides were obtained from Sigma Life Science (Sapporo, Japan) or Scrum, Inc. (Tokyo, Japan). ELISA was used to measure in vitro reactions between synthetic peptides and G2 as described previously . For normal ELISA, 96‐well plates (Nunc, Roskilde, Denmark) were coated with 100 ng/well of Ag peptides in 50 μl PBS (pH 7.0).…”

Section: Methodsmentioning

confidence: 99%

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A multispecific monoclonal antibody G2 recognizes at least three completely different epitope sequences with high affinity

et al. 2017

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A monoclonal antibody (mAb) G2 possesses an unusual characteristic of reacting with at least three proteins (ATP6V1C1, SEPT3, and C6H10orf76) other than its original antigen, chicken prion protein (ChPrP). The epitopes on ChPrP and ATP6V1C1 have been identified previously. In this study, we identified the epitope in the third protein, SEPT3. Interestingly, there was no amino acid sequence similarity among the epitopes on the three proteins. These epitopes had high binding affinities to G2 (K = ∼10 M for monovalent binding and K = ∼10 M for divalent binding), as determined using a SPR biosensor. This is the first report on a three-in-one mAb recognizing completely different epitope sequences with high affinity. Additionally, competitive ELISA indicated that the binding sites on G2, specific for the three different epitopes, overlapped, suggesting that the antigen-binding site may be flexible in the free form and capable of adapting to at least three different conformations to enable interactions with three different antigens.

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Molecular characterization of chicken prion proteins by C-terminal-specific monoclonal antibodies

Cited by 9 publications

References 12 publications

Identification and characterization of a multispecific monoclonal antibody G2 against chicken prion protein

Identification and characterization of a multispecific monoclonal antibody G2 against chicken prion protein

Distribution of the cellular prion protein in the central nervous system of the chicken

A multispecific monoclonal antibody G2 recognizes at least three completely different epitope sequences with high affinity

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