Ukr.Biochem.J. 2023; Volume 95, Issue 1, Jan-Feb, pp. 20-30


Effect of trifluoroethanol on antibodies binding properties

S. A. Bobrovnik*, M. O. Demchenko, S. V. Komisarenko

Department of Molecular Immunology, Palladin Institute of Biochemistry,
National Academy of Sciences of Ukraine, Kyiv;

Received: 01 December 2022; Revised: 22 February 2023;
Accepted: 13 April 2023; Available on-line: 27 April 2023

The studies on the influence of organic co-solvents on the structure and function of antibodies are of key interest, especially in view of antibodies broad use as recognizing elements in different analytical systems. Here we studied the effect of co-solvent 2,2,2-trifluoroethanol (TFE) on the ability of anti-ovalbumin monoclonal antibodies to interact with its specific antigen. Antibody affinity to antigen and the rate constants of antibody binding to immobilized antigen were analyzed. Changes in antibody reactivity with incubation time which depended on TFE concentration and temperature were revealed. When treatment of antibodies with TFE was carried out at 0°C, we observed nonlinear, non-monotonous changes of antibody reactivity with initial fast decrease and substantial increase as incubation continued that may be related to the loss of antigen binding reactivity by some part of antibodies at the start but its restoration when the incubation proceeds.

Keywords: , , , ,


  1. Sharma S, Byrne H, O’Kennedy RJ. Antibodies and antibody-derived analytical biosensors. Essays Biochem. 2016;60(1):9-18. PubMed, PubMedCentral, CrossRef
  2. Farka Z, Juřík T, Kovář D, Trnková L, Skládal P. Nanoparticle-Based Immunochemical Biosensors and Assays: Recent Advances and Challenges. Chem Rev. 2017;117(15):9973-10042. PubMed, CrossRef
  3. Liu M, Xu M, Loh XJ, Abe H, Tsumuraya T, Fujii I, Li J, Son TI, Ito Y. PEGylated antibody in organic media. J Biosci Bioeng. 2011;111(5):564-568. PubMed, CrossRef
  4. Chapman AP. PEGylated antibodies and antibody fragments for improved therapy: a review. Adv Drug Deliv Rev. 2002;54(4):531-545. PubMed, CrossRef
  5. Hermanson GT. Bioconjugate techniques. Academic press, 2013. CrossRef
  6. Rehan M, Younus H. Effect of organic solvents on the conformation and interaction of catalase and anticatalase antibodies. Int J Biol Macromol. 2006;38(3-5):289-295. PubMed, CrossRef
  7. Buck M. Trifluoroethanol and colleagues: cosolvents come of age. Recent studies with peptides and proteins. Q Rev Biophys. 1998;31(3):297-355. PubMed, CrossRef
  8. Gast K, Zirwer D, Müller-Frohne M, Damaschun G. Trifluoroethanol-induced conformational transitions of proteins: insights gained from the differences between alpha-lactalbumin and ribonuclease A. Protein Sci. 1999;8(3):625-634. PubMed, PubMedCentral, CrossRef
  9. Carre B, Devynck J. The acidity functions of trifluoroethanol and hexafluoroisopropanol, and their mixtures with water. Anal Chim Acta. 1981;131:141-147. CrossRef
  10. Reichardt C, Welton T. Solvents and solvent effects in organic chemistry. John Wiley & Sons, 2011. CrossRef
  11. Sönnichsen FD, Van Eyk JE, Hodges RS, Sykes BD. Effect of trifluoroethanol on protein secondary structure: an NMR and CD study using a synthetic actin peptide. Biochemistry. 1992;31(37):8790-8798. PubMed, CrossRef
  12. Luo P, Baldwin RL. Mechanism of helix induction by trifluoroethanol: a framework for extrapolating the helix-forming properties of peptides from trifluoroethanol/water mixtures back to water. Biochemistry. 1997;36(27):8413-8421. PubMed, CrossRef
  13. Santiveri CM, Pantoja‐Uceda D, Rico M, Jimenez MA. Beta-hairpin formation in aqueous solution and in the presence of trifluoroethanol: a (1)H and (13)C nuclear magnetic resonance conformational study of designed peptides. Biopolymers. 2005;79(3):150-162. PubMed, CrossRef
  14. Cammers-Goodwin A, Allen TJ, Oslick SL, McClure KF, Lee JH, Kemp DS. Mechanism of stabilization of helical conformations of polypeptides by water containing trifluoroethanol. J Am Chem Soc. 1996;118(13):3082-3090. CrossRef
  15. Roccatano D, Colombo G, Fioroni M, Mark AE. Mechanism by which 2,2,2-trifluoroethanol/water mixtures stabilize secondary-structure formation in peptides: a molecular dynamics study. Proc Natl Acad Sci USA. 2002;99(19):12179-12184. PubMed, PubMedCentral, CrossRef
  16. Dave S, Mahajan S, Chandra V, Gupta P. Trifluoroethanol stabilizes the molten globule state and induces non-amyloidic turbidity in stem bromelain near its isoelectric point. Int J Biol Macromol. 2011;49(4):536-542. PubMed, CrossRef
  17. Hirota N, Mizuno K, Goto Y. Cooperative alpha-helix formation of beta-lactoglobulin and melittin induced by hexafluoroisopropanol. Protein Sci. 1997;6(2):416-421. PubMed, PubMedCentral, CrossRef
  18. Sen P, Ahmad B, Rabbani G, Khan RH. 2,2,2-Trifluroethanol induces simultaneous increase in alpha-helicity and aggregation in alkaline unfolded state of bovine serum albumin. Int J Biol Macromol. 2010;46(2):250-254. PubMed, CrossRef
  19. Anderson VL, Ramlall TF, Rospigliosi CC, Webb WW, Eliezer D. Identification of a helical intermediate in trifluoroethanol-induced alpha-synuclein aggregation. Proc Natl Acad Sci USA. 2010;107(44):18850-18855. PubMed, PubMedCentral, CrossRef
  20. Anderson VL, Webb WW. A desolvation model for trifluoroethanol-induced aggregation of enhanced green fluorescent protein. Biophys J. 2012;102(4):897-906. PubMed, PubMedCentral, CrossRef
  21. Shanmugam G, Reddy SMM, Natarajan V, Madhan B. 2,2,2-Trifluoroethanol disrupts the triple helical structure and self-association of type I collagen. Int J Biol Macromol. 2013;54:155-159. PubMed, CrossRef
  22. Gupta P, Deep S. Intermediate conformation between native β-sheet and non-native α-helix is a precursor of trifluoroethanol-induced aggregation of human carbonic anhydrase-II. Biochem Biophys Res Commun. 2014;449(1):126-131. PubMed, CrossRef
  23. Friguet B, Chaffotte AF, Djavadi-Ohaniance L, Goldberg ME. Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. J Immunol Methods. 1985;77(2):305-319. PubMed, CrossRef
  24. Stevens FJ. Modification of an ELISA-based procedure for affinity determination: correction necessary for use with bivalent antibody. Mol Immunol. 1987;24(10):1055-1060. PubMed, CrossRef
  25. Bobrovnik SA. Determination of antibody affinity by ELISA. Theory. J Biochem Biophys Methods. 2003;57(3):213-236. PubMed, CrossRef
  26. Nygren H, Czerkinsky C, Stenberg M. Dissociation of antibodies bound to surface-immobilized antigen. J Immunol Methods. 1985;85(1):87-95. PubMed, CrossRef
  27. Nygren H, Werthen M, Stenberg M. Kinetics of antibody binding to solid-phase-immobilised antigen. Effect of diffusion rate limitation and steric interaction. J Immunol Methods. 1987;101(1):63-71. PubMed, CrossRef
  28. Bobrovnik SA. Dynamics of the interaction of monoclonal antibodies with antigens immobilized on plates. Ukr Biokhim Zhurn. 1998;70(3):118-128. (In Russian). PubMed
  29. Cornish-Bowden A. Fundamentals of enzyme kinetics. Weinheim, Germany: Wiley-Blackwell, 2012. 510 p.
  30. Bobrovnik SA. Determination of kinetic parameters for both reversible and irreversible first-order reactions. J Biochem Biophys Methods. 1998;37(1-2):53-68. PubMed, CrossRef
  31. Bobrovnik SA. Polyreactive immunoglobulins: molecular properties and functions. Comments Molec Cell Biophys. 1999;9:323-356.
  32. Bobrovnik SA, Demchenko MO, Komisarenko SV. Effect of trifluoroethanol on antibody reactivity against corresponding and nonrelated antigens. Ukr Biochem J. 2018;90(4):80-89. CrossRef

Creative CommonsThis work is licensed under a Creative Commons Attribution 4.0 International License.