Structural and functional study of mouse antidigoxin monoclonal antibody against thermal variation.
Conformation, heat-induced conformational changes, and the stability of mouse monoclonal antibody against digoxin were analyzed by circular dichroism (CD), fluorescence, UV-Vis spectroscopy, and ELISA techniques. Since both biological function and aggregation depend on the conformational properties of mouse IgG molecules, a series of sensitive and rapid physical methods were introduced to assess conformational changes and structural stability as a function of temperature. CD spectra of IgG show the characteristics expected for beta-proteins. Denaturation experiments performed through optical spectroscopic methods and affinity measurement indicated that the antibody unfolds cooperatively in a single transition. Thus, unfolding/refolding equilibrium proceeds via a simple two-state mechanism (N equilibrium U), where only the native and the unfolded states are significantly populated. Thermal-induced denaturation, however, is not completely reversible, and the partial loss of binding capacity might be due, at least in part, to incorrect refolding of the long loops (CDRs), which are responsible for antigen recognition. The results show that purified IgG is temperature stable. Of course, affinity assessment as measured by ELISA indicated that increasing the temperature decreases the affinity of IgG toward digoxin.
Kashanian
Soheila
S
Department of Chemistry, Faculty of Science, Razi University, Kermanshah, Iran. kashanian_s@yahoo.com
Ghobadi
Sirous
S
Omidfar
Kobra
K
Ravan
Hadi
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D000915:Antibody Affinity; D002942:Circular Dichroism; D004077:Digoxin / Q000276:immunology*; D006358:Hot Temperature; D051379:Mice; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013696:Temperature
D000911:Antibodies, Monoclonal; D004077:Digoxin