Effects of substitutions of amino acids on the thermal stability of the Fv fragments of antibodies.

The thermal stability of Fv fragments was examined by circular dichroism (CD) spectrometry and high-performance liquid chromatography. We analyzed three Fv fragments: that of a monoclonal antibody D1.3 and two derivatives of it. After separation of wild-type VH and VL fragments, thermal denaturation of each fragment was monitored by CD spectrometry. The results indicated that the dissociation of Fv into VH and VL fragments seemed to be coupled with the denaturation of each fragment and that the thermal denaturation of VH and VL fragments was prevented when they were associated with one another. The analysis of the three Fv fragments also indicated that, in some cases, differences in amino acids even within the CDRs could have significant effects on the thermal stability of the complex between VH and VL fragments.