Influence of antibody-antigen interaction on fluorescence of Rhodamine 6G conjugated to the antibodies measured in human serum

Fluorescent dyes conjugated to monoclonal antibodies specifically trastuzumab (TRAS) and pertuzumab (PERT) serve as sensitive reporters of changes in the local energetic environment arising from antibody-antibody and antibody-antigen interactions. Upon covalent conjugation, the antibody local environment perturbates the energetic state of the dye, resulting in changes of the fluorescence emission energy and emission width of the dye. The dependence of these spectral parameters on antibody concentration was measured in HBS-8, diluted and undiluted human serum. In addition, the emission energy and emission width of dye-conjugated TRAS were investigated as a function of recombinant human epidermal growth factor receptor 2 (HER2) extracellular domain concentration in HBS-8 and in diluted and undiluted human serum. Antibody-antibody and antibody-antigen interactions produce remarkable contributions into the emission energy and emission width. These contributions depend on the composition of the surrounding medium. Our study showed that the samples containing 100 nM TRAS in serum exhibited the largest spectral changes upon interaction with HER2 suggesting that antibody-antigen interaction are not linear across concentration range of the antibodies. Moreover, our results demonstrate that interactions between dye-conjugated antibodies and their binding partners can be detected under physiologically relevant conditions. These findings establish fluorescence emission energy and emission width analysis as a potential approach for probing antibody interactions in complex biological matrices.