A comparative study of capillary electrophoresis and isothermal titration calorimetry for the determination of binding constant of human serum albumin to monoclonal antibody

• Published in: Electrophoresis Vol. 36; no. 11-12; pp. 1274 - 1281
• Main Authors: Andrási, Melinda, Lehoczki, Gábor, Nagy, Zoltán, Gyémánt, Gyöngyi, Pungor, András, Gáspár, Attila
• Format: Journal Article
• Language: English
• Published: Germany Blackwell Publishing Ltd 01-06-2015
• Subjects: ACE; Antibodies, Monoclonal - immunology; Antigens; Binding; Binding constant; Binding sites; Binding Sites, Antibody; Calorimetry - methods; Constants; CZE; Electrophoresis; Electrophoresis, Capillary - methods; HSA; Human; Humans; mAb; Plotting; Serum Albumin - immunology; Titration calorimetry; ACE; Binding constant; HSA; CZE; mAb
• ISSN: 0173-0835; 1522-2683
• DOI: 10.1002/elps.201400513

This paper focuses on the investigation of the interactions between the anti‐HSA‐mAb and its protein antigen using CZE, ACE, and isothermal titration calorimetry. The CZE revealed the formation of the anti‐HSA‐mAb·HSA and anti‐HSA‐mAb·(HSA)2 complexes and the binding constants determined by plotting the amount of the bound anti‐HSA‐mAb as a function of the concentration of HSA. The ACE provided information on the binding strength from the change in effective electrophoretic mobility of the anti‐HSA‐mAb. These two separation techniques estimated the presence of two binding sites. The equilibrium dissociation constant values obtained by CZE and ACE were found to be 2.26 × 10−6 M for anti‐HSA‐mAb·HSA, 1.22 × 10−6 M for anti‐HSA‐mAb·(HSA)2 and 4.45 × 10−8 M for anti‐HSA‐mAb·HSA, 1.08 × 10−7 M for anti‐HSA‐mAb·(HSA)2, respectively. The dissociation constant data obtained by ACE were in congruence with the values obtained by isothermal titration calorimetry (2.74 × 10−8 M, 1.04 × 10−7 M).