Heat denaturation of the antibody, a multi-domain protein

Abstract

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.

Heat inactivation of serum interferes with the immunoanalysis of antibodies to SARS-CoV-2

“…16,17 Previous studies have shown that antibodies can be denatured and lose their antigen binding activities after heating, 18 and IgM is reported to be less thermally stable than IgG 19,20 due to their different compositions and structures of heavy chains. 21 This is consistent with our results that SARS-CoV-2 IgM concentration decreased more significantly than IgG after heating. In .…”

Heat inactivation of serum interferes with the immunoanalysis of antibodies to SARS‐CoV‐2

“…The decrease in SARS‐CoV‐2 antibody levels may be related to their structural change in denaturation and aggregation 16,17 . Previous studies have shown that antibodies can be denatured and lose their antigen‐binding activities after heating, 18 and IgM is reported to be less thermally stable than IgG 19,20 due to their different compositions and structures of heavy chains 21 . This is consistent with our results that SARS‐CoV‐2 IgM concentration decreased more significantly than IgG after heating.…”

“…20 due to their different compositions and structures of heavy chains 21. This is consistent with our results that SARS-CoV-2 IgM concentration decreased more significantly than IgG after heating.…”

Rituximab-specific DNA aptamers are able to selectively recognize heat-treated antibodies

“…DLS analyses revealed a tendency for oligomerization after 5 min at 70˚C, while a mix of mono-and oligomers was observed after 1 min at 80˚C. In line with the multi-domain aggregation study of antibodies, we conclude that prolonged heating at 80˚C leads to further aggregation and finally precipitation of the antibody [6,7]. It is noteworthy to mention that during aptamer screening rituximab was consequently bound to protein A, which prevented the formation of aggregates by keeping the protein molecules spatially separated [7].…”