Abstract
Antibodies can be extremely useful tools for the field of triplet repeats diseases. These reagents are important for localizing proteins in tissues and they can be used in the isolation and characterization of the components of protein complexes. In the context of huntingtin (Htt), antibodies can distinguish Htt with normal or an expanded polyglutamine (polyQ) repeats, and they can identify distinct conformations of Htt. Htt is the protein that, when mutated to contain an expanded polyQ motif, causes Huntington’s disease (HD). Our group has produced monoclonal and recombinant single-chain antibodies (intrabodies) that can be used for these purposes and to perturb the function of Htt in living cells. Studies with anti-Htt intrabodies have led to identification of novel pathogenic epitopes. Moreover, some of the isolated intrabodies can reduce the neurotoxicity of mutant Htt in cell culture and animal models of HD. Thus, the production of antibodies and intrabodies has made a significant contribution to the understanding of HD pathogenesis and has introduced a novel strategy to treat this debilitating neurodegenerative disorder.
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References
Bennett MJ, Huey-Tubman KE, Herr AB et al (2002) A linear lattice model for polyglutamine in CAG-expansion diseases. Proc Natl Acad Sci USA 99:11634–11639
Ko J, Ou S, Patterson PH (2001) New anti-huntingtin monoclonal antibodies: implications for huntingtin conformation and its binding proteins. Brain Res Bull 56:319–329
Ko J, Patterson PH Unpublished data
Khoshnan A, Ko J, Patterson PH (2002) Effects of intracellular expression of anti-huntingtin antibodies of various specificities on mutant huntingtin aggregation and toxicity. Proc Natl Acad Sci USA 99:1002–1007
Jackson JR, Salecker I, Dong X et al (1998) Polyglutamine-expanded human huntingtin transgenes induce degeneration of Drosophila photoreceptor neurons. Neuron 21:633–642
Jackson G, Khoshnan A, Patterson PH Unpublished data
Southwell AL, Khoshnan A, Dunn DE et al (2008) Intrabodies binding to proline-rich domains of mutant huntingtin increase its turnover and reduce neurotoxicity. J Neurosci 28:9013–9020
Legleiter J, Lotz GP, Miller J et al (2009) Monoclonal antibodies recognize distinct conformational epitopes formed by polyglutamine in a mutant huntingtin fragment. J Biol Chem 284:21647–21658
Southwell AL, Ko J, Patterson PH (2009) Intrabody gene therapy ameliorates motor, cognitive, and neuropathological symptoms in multiple mouse models of Huntington’s disease. J Neurosci 29:13589–13602
Colby DW, Chu Y, Cassady JP et al (2004) Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody. Proc Natl Acad Sci USA 101:17616–17621
Snyder-Keller A, McLear JA, Hathorn T et al (2010) Early or late-stage anti-N-terminal huntingtin intrabody gene therapy reduces pathological features in B6.HDR6/1 mice. J Neuropathol Exp Neurol 69:1078–1085
Wang CE, Zhou H, McGuire JR et al (2008) Suppression of neuropil aggregates and neurological symptoms by an intracellular antibody implicates the cytoplasmic toxicity of mutant huntingtin. J Cell Biol 181:803–816
Khoshnan A, Ko J, Watkin EE et al (2004) Activation of the IkappaB kinase complex and nuclear factor-kappaB contributes to mutant huntingtin neurotoxicity. J Neurosci 24:7999–8008
Harlow E, Lane D (1988) Antibodies: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 55–56 and 72–73
Gullick WJ (1988) Production of antiserum to synthetic peptides. Methods Mol Biol 3:341–354
Hawkes R, Niday E, Gordan J (1982) A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem 119:142–147
Al Moudallal Z, Altschuh D, Briand JP et al (1984) Comparative sensitivity of different ELISA procedures for detecting monoclonal antibodies. J Immunol Methods 68:35–43
Birk H-W, Koepsell H (1987) Reaction of monoclonal antibodies with plasma membrane proteins after binding on nitrocellulose: renaturation of antigenic binding sites and reduction of nonspecific antibody binding. Anal Biochem 164:12–22
Davies DR, Padlan EA, Sheriff S (1990) Antibody-antigen complexes. Annu Rev Biochem 59:439–473
Ou SK, Patterson PH (1997) A more efficient and economical approach for monoclonal antibody production. J Immunol Methods 209:105–108
Stang BV, Wood PA, Reddington JJ et al (1998) Monoclonal antibody production in gas-permeable flexible flasks, using serum-free medium. Contemp Top Lab Anim Sci 37:55–60
Scott LE, Aggett H, Glencross DK (2001) Manufacture of pure monoclonal antibodies by heterogeneous culture without downstream purification. Biotechnique 31:666–668
Jackson LR, Trudel LJ, Lipman NS (1999) Small-scale monoclonal antibody production in vitro: methods and resources. Lab Anim 28:20–30
Marx U, Embleton MJ, Fischer R et al (1997) Monoclonal antibody production. The report and recommendations of ECVAM workshop 23. Altern Lab Anim 25:121–137
Ou SK, Hwang JM, Patterson PH (1993) A modified method for obtaining large amounts of high titer polyclonal ascites fluid. J Immunol Methods 165:75–80
Rondon IJ, Marasco WA (1997) Intracellular antibodies (intrabodies) for gene therapy of infectious diseases. Annu Rev Microbiol 51:257–283
Lecerf J-M, Shirley TL, Zhu Q et al (2001) Human single chain Fv intrabodies counteract in situ huntingtin aggregation in cellular models of Huntington’s disease. Proc Natl Acad Sci USA 98:4764–4769
Winter G (1998) Making antibody and peptide ligands by repertoire selection technologies. J Mol Recognit 11:126–127
Bradbury AR, Sidhu S, Dübel S et al (2011) Beyond natural antibodies: the power of in vitro display technologies. Nat Biotechnol 29:245–254
Colby DW, Kellogg BA, Graff CP et al (2004) Engineering antibody affinity by yeast surface display. Methods Enzymol 388:348–358
Bidlingmaier S, Liu B (2011) Construction of yeast surface-displayed cDNA libraries. Methods Mol Biol 729:199–210
Matthew WD, Sandrock AW (1987) Cyclophosphamide treatment used to manipulate the immune response for the production of monoclonal antibodies. J Immunol Methods 100:73–82
Lebron JA, Shen H, Bjorkman PJ et al (1999) Tolerization of adult mice to immunodominant proteins before monoclonal antibody production. J Immunol Methods 222:59–63
Lipman NS, Trudel LJ, Murphy JC et al (1992) Comparison of immune response potentiation and in vivo inflammatory effects of Freund’s and RIBI adjuvants in mice. Lab Anim Sci 42:193–197
Rudbach JA, Cantrell JL, Ulrich JT (1988) Molecularly engineered microbial immunostimulators. In: Lasky L (ed) Technological advances in vaccine development. Alan R. Liss, New York, pp 443–454
Acknowledgment
Work cited from the authors’ laboratory was supported by the Hereditary Disease Foundation.
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Khoshnan, A., Ou, S., Ko, J., Patterson, P.H. (2013). Antibodies and Intrabodies Against Huntingtin: Production and Screening of Monoclonals and Single-Chain Recombinant Forms. In: Kohwi, Y., McMurray, C. (eds) Trinucleotide Repeat Protocols. Methods in Molecular Biology, vol 1010. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-411-1_15
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DOI: https://doi.org/10.1007/978-1-62703-411-1_15
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