Structural and functional study of rabbit polyclonal antibody for immunoassay purposes.
In this study, we investigated the effects of some denaturants, such as urea and heat, on structure and function of rabbit polyclonal antibody and its Fab fragments. Thermal unfolding studies by circular dichroism of antibody and Fab fragments showed that in acidic pH, antibody has multi-transitions whereas Fab fragments have one transition curve; however in neutral pH, thermal unfolding of both had one transition. Effects of urea on the structure of antibody and Fab were studied through fluorescence spectroscopy. Despite exposure of protein to high concentration of denaturant, partial unfolding occurred in both antibody and Fab, but the denaturation of Fab was more considerable than that of antibody. Functional studies indicated that urea and heat causes a decrease in affinity in both antibody and Fab, but deactivation of Fab is more considerable in comparison with the antibody molecule. Turbidity study of antibody and Fab showed that aggregation of Fab occurred at lower temperatures than that of antibody. Our results indicate that Fab has higher sensitivity in comparison with antibody in the unfolding, deactivation, and aggregation processes. Therefore, our data proposes a stabilizing role for Fc.
Kashanian
Soheila
S
Department of Chemistry and Nanoscience & Nanotechnology Research Center (NNRC), Faculty of Science, Razi University, Kermanshah, Iran. kashanian_s@yahoo.com
Ravan
Hadi
H
Ghobadi
Sirous
S
Omidfar
Kobra
K
Askari
Soyebeh
S
D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000276:immunology; D002942:Circular Dichroism; D006358:Hot Temperature; D007118:Immunoassay; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000276:immunology; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000276:immunology; D017510:Protein Folding; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000737:chemistry
D000906:Antibodies; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D014508:Urea