Biophysical Reviews

, Volume 10, Issue 2, pp 255–258 | Cite as

Heat denaturation of the antibody, a multi-domain protein

  • Yoko Akazawa-Ogawa
  • Hidenori Nagai
  • Yoshihisa Hagihara


The antibody is one of the most well-studied multi-domain proteins because of its abundance and physiological importance. In this article, we describe the effect of the complex, multi-domain structure of the antibody on its denaturation by heat. Natural antibodies are composed of 6 to 70 immunoglobulin fold domains, and are irreversibly denatured at high temperatures. Although the separated single immunoglobulin fold domain can be refolded after heat denaturation, denaturation of pairs of such domains is irreversible. Each antibody subclass exhibits a distinct heat tolerance, and IgE is especially known to be heat-labile. IgE starts unfolding at a lower temperature compared to other antibodies, because of the low stability of its CH3 domain. Each immunoglobulin domain starts unfolding at different temperatures. For instance, the CH3 domain of IgG unfolds at a higher temperature than its CH2 domain. Thus, the antibody has a mixture of folded and unfolded structures at a certain temperature. Co-existence of these folded and unfolded domains in a single polypeptide chain may increase the tendency to aggregate which causes the inactivation of the antibody.


Antibody Heat denaturation Aggregation Protein stability Single-domain antibody 


Compliance with ethical standards

Conflict of interest

Yoko Akazawa-Ogawa declares that she has no conflict of interest. Hidenori Nagai declares that he has no conflict of interest. Yoshihisa Hagihara declares that he has no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.


  1. Akazawa-Ogawa Y, Takashima M, Lee YH, Ikegami T, Goto Y, Uegaki K, Hagihara Y (2014) Heat-induced irreversible denaturation of the camelid single domain VHH antibody is governed by chemical modifications. J Biol Chem 289:15666–15679CrossRefPubMedPubMedCentralGoogle Scholar
  2. Akazawa-Ogawa Y, Uegaki K, Hagihara Y (2016) The role of intra-domain disulfide bonds in heat-induced irreversible denaturation of camelid single domain VHH antibodies. J Biochem 159:111–121CrossRefPubMedGoogle Scholar
  3. Augener W, Grey HM (1970) Studies on the mechanism of heat aggregation of human γG. J Immunol 105:1024–1030PubMedGoogle Scholar
  4. Binaghi RA, Demeulemester C (1983) Influence of the medium on the heat and acid denaturation of IgE. J Immunol Methods 65:225–233CrossRefPubMedGoogle Scholar
  5. Bloch KJ, Morse HC 3rd, Austen KF (1968) Biologic properties of rat antibodies. I. Antigen-binding by four classes of anti-DNP antibodies. J Immunol 101:650–657PubMedGoogle Scholar
  6. Brader ML, Estey T, Bai S, Alston RW, Lucas KK, Lantz S, Landsman P, Maloney KM (2015) Examination of thermal unfolding and aggregation profiles of a series of developable therapeutic monoclonal antibodies. Mol Pharm 12:1005–1017CrossRefPubMedGoogle Scholar
  7. Chen W, Kong L, Connelly S, Dendle JM, Liu Y, Wilson IA, Powers ET, Kelly JW (2016) Stabilizing the CH2 domain of an antibody by engineering in an enhanced aromatic sequon. ACS Chem Biol 11:1852–1861CrossRefPubMedPubMedCentralGoogle Scholar
  8. Demarest SJ, Rogers J, Hansen G (2004) Optimization of the antibody C(H)3 domain by residue frequency analysis of IgG sequences. J Mol Biol 335:41–48CrossRefPubMedGoogle Scholar
  9. Demarest SJ, Hopp J, Chung J, Hathaway K, Mertsching E, Cao X, George J, Miatkowski K, LaBarre MJ, Shields M, Kehry MR (2006) An intermediate pH unfolding transition abrogates the ability of IgE to interact with its high affinity receptor FcεRIα. J Biol Chem 281:30755–30767CrossRefPubMedGoogle Scholar
  10. Deutscher SL, Crider ME, Ringbauer JA, Komissarov AA, Quinn TP (1996) Stability studies of nucleic acid-binding Fab isolated from combinatorial bacteriophage display libraries. Arch Biochem Biophys 333:207–213CrossRefPubMedGoogle Scholar
  11. Feige MJ, Walter S, Buchner J (2004) Folding mechanism of the CH2 antibody domain. J Mol Biol 344:107–118CrossRefPubMedGoogle Scholar
  12. Feige MJ, Groscurth S, Marcinowski M, Shimizu Y, Kessler H, Hendershot LM, Buchner J (2009) An unfolded CH1 domain controls the assembly and secretion of IgG antibodies. Mol Cell 34:569–579CrossRefPubMedPubMedCentralGoogle Scholar
  13. Garber E, Demarest SJ (2007) A broad range of Fab stabilities within a host of therapeutic IgGs. Biochem Biophys Res Commun 355:751–757CrossRefPubMedGoogle Scholar
  14. Ghirlando R, Lund J, Goodall M, Jefferis R (1999) Glycosylation of human IgG-fc: influences on structure revealed by differential scanning micro-calorimetry. Immunol Lett 68:47–52CrossRefPubMedGoogle Scholar
  15. Hagihara Y, Shiraki K, Nakamura T, Uegaki K, Takagi M, Imanaka T, Yumoto N (2002) Screening for stable mutants with amino acid pairs substituted for the disulfide bond between residues 14 and 38 of bovine pancreatic trypsin inhibitor (BPTI). J Biol Chem 277:51043–51048CrossRefPubMedGoogle Scholar
  16. Hagihara Y, Matsuda T, Yumoto N (2005) Cellular quality control screening to identify amino acid pairs for substituting the disulfide bonds in immunoglobulin fold domains. J Biol Chem 280:24752–24758CrossRefPubMedGoogle Scholar
  17. Henry AJ, McDonnell JM, Ghirlando R, Sutton BJ, Gould HJ (2000) Conformation of the isolated Cε3 domain of IgE and its complex with the high-affinity receptor, FcεRI. Biochemistry 39:7406–7413CrossRefPubMedGoogle Scholar
  18. Holdom MD, Davies AM, Nettleship JE, Bagby SC, Dhaliwal B, Girardi E, Hunt J, Gould HJ, Beavil AJ, McDonnell JM, Owens RJ, Sutton BJ (2011) Conformational changes in IgE contribute to its uniquely slow dissociation rate from receptor FcεRI. Nat Struct Mol Biol 18:571–576CrossRefPubMedPubMedCentralGoogle Scholar
  19. Hunt J, Beavil RL, Calvert RA, Gould HJ, Sutton BJ, Beavil AJ (2005) Disulfide linkage controls the affinity and stoichiometry of IgE Fcε3–4 binding to FcεRI. J Biol Chem 280:16808–16814CrossRefPubMedGoogle Scholar
  20. Indyk HE, Williams JW, Patel HA (2008) Analysis of denaturation of bovine IgG by heat and high pressure using an optical biosensor. Int Dairy J 18:359–366CrossRefGoogle Scholar
  21. Ishizaka K, Ishizaka T, Menzel AE (1967) Physicochemical properties of reaginic antibody. VI. Effect of heat on gamma-E-, gamma-G- and gamma-A-antibodies in the sera of ragweed sensitive patients. J Immunol 99:610–618PubMedGoogle Scholar
  22. Ishizaka T, Helm B, Hakimi J, Niebyl J, Ishizaka K, Gould H (1986) Biological properties of a recombinant human immunoglobulin epsilon-chain fragment. Proc Natl Acad Sci U S A 83:8323–8327CrossRefPubMedPubMedCentralGoogle Scholar
  23. Ito T, Tsumoto K (2013) Effects of subclass change on the structural stability of chimeric, humanized, and human antibodies under thermal stress. Protein Sci 22:1542–1551CrossRefPubMedPubMedCentralGoogle Scholar
  24. Jespers L, Schon O, Famm K, Winter G (2004a) Aggregation-resistant domain antibodies selected on phage by heat denaturation. Nat Biotechnol 22:1161–1165CrossRefPubMedGoogle Scholar
  25. Jespers L, Schon O, James LC, Veprintsev D, Winter G (2004b) Crystal structure of HEL4, a soluble, refoldable human V(H) single domain with a germ-line scaffold. J Mol Biol 337:893–903CrossRefPubMedGoogle Scholar
  26. Kellogg DE, Rybalkin I, Chen S, Mukhamedova N, Vlasik T, Siebert PD, Chenchik A (1994) TaqStart Antibody: “hot start” PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase. BioTechniques 16:1134–1137PubMedGoogle Scholar
  27. Ladenson RC, Crimmins DL, Landt Y, Ladenson JH (2006) Isolation and characterization of a thermally stable recombinant anti-caffeine heavy-chain antibody fragment. Anal Chem 78:4501–4508CrossRefPubMedGoogle Scholar
  28. Mainer G, Sanchez L, Ena JM, Calvo M (1997) Kinetic and thermodynamic parameters for heat denaturation of bovine milk IgG, IgA and IgM. J Food Sci 62:1034–1038CrossRefGoogle Scholar
  29. Mainer G, Domínguez E, Randrup M, Sánchez L, Calvo M (1999) Effect of heat treatment on anti-rotavirus activity of bovine colostrum. J Dairy Res 66:131–137CrossRefPubMedGoogle Scholar
  30. Martsev SP, Chumanevich AA, Vlasov AP, Dubnovitsky AP, Tsybovsky YI, Deyev SM, Cozzi A, Arosio P, Kravchuk ZI (2000) Antiferritin single-chain Fv fragment is a functional protein with properties of a partially structured state: comparison with the completely folded V(L) domain. Biochemistry 39:8047–8057CrossRefPubMedGoogle Scholar
  31. Mizuguchi H, Nakatsuji M, Fujiwara S, Takagi M, Imanaka T (1999) Characterization and application to hot start PCR of neutralizing monoclonal antibodies against KOD DNA polymerase. J Biochem 126:762–768CrossRefPubMedGoogle Scholar
  32. Prouvost-Danon A, Binaghi RA, Abadie A (1977) Effect of heating at 56 °C on mouse IgE antibodies. Immunochemistry 14:81–84CrossRefPubMedGoogle Scholar
  33. Sharkey DJ, Scalice ER, Christy KG, Atwood SM, Daiss JL (1994) Antibodies as thermolabile switches: high temperature triggering for the polymerase chain reaction. Biotechnology 12:506–509CrossRefPubMedGoogle Scholar
  34. van der Linden RH, Frenken LG, de Geus B, Harmsen MM, Ruuls RC, Stok W, de Ron L, Wilson S, Davis P, Verrips CT (1999) Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies. Biochim Biophys Acta 1431:37–46CrossRefPubMedGoogle Scholar
  35. Vermeer AW, Norde W (2000) The thermal stability of immunoglobulin: unfolding and aggregation of a multi-domain protein. Biophys J 78:394–404CrossRefPubMedPubMedCentralGoogle Scholar
  36. Vermeer AW, Norde W, van Amerongen A (2000) The unfolding/denaturation of immunogammaglobulin of isotype 2b and its F(ab) and F(c) fragments. Biophys J 79:2150–2154CrossRefPubMedPubMedCentralGoogle Scholar
  37. Voynov V, Chennamsetty N, Kayser V, Helk B, Forrer K, Zhang H, Fritsch C, Heine H, Trout BL (2009) Dynamic fluctuations of protein–carbohydrate interactions promote protein aggregation. PLoS One 4:e8425CrossRefPubMedPubMedCentralGoogle Scholar
  38. Wurzburg BA, Garman SC, Jardetzky TS (2000) Structure of the human IgE-Fc Cε3-Cε4 reveals conformational flexibility in the antibody effector domains. Immunity 13:375–385CrossRefPubMedGoogle Scholar
  39. Zheng K, Bantog C, Bayer R (2011) The impact of glycosylation on monoclonal antibody conformation and stability. MAbs 3:568–576CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag GmbH Germany, part of Springer Nature 2017

Authors and Affiliations

  1. 1.Biomedical Research InstituteNational Institute of Advanced Industrial Science and Technology (AIST)IkedaJapan
  2. 2.AIST-Osaka University Advanced Photonics and Biosensing Open Innovation LaboratoryNational Institute of Advanced Industrial Science and Technology (AIST)SuitaJapan

Personalised recommendations