The James B Munro research team at the University of Massachusetts Medical School used fluorescence correlation spectroscopy to... Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects cells through binding to angiotensin-converting enzyme 2 (ACE2). This interaction is mediated by the receptor-binding domain (RBD) of the viral spike (S) glycoprotein. Structural and dynamic data have shown that S can adopt multiple conformations, which controls the exposure of the ACE2-binding site in the RBD. Here, using single-molecule Förster resonance energy transfer (smFRET) imaging, we report the effects of ACE2 and antibody binding on the conformational dynamics of S from the Wuhan-1 strain and in the presence of the D614G mutation. We find that D614G modulates the energetics of the RBD position in a manner similar to ACE2 binding. We also find that antibodies that target diverse epitopes, including those distal to the RBD, stabilize the RBD in a position competent for ACE2 binding. Parallel solution-based binding experiments using fluorescence correlation spectroscopy (FCS) indicate antibody-mediated enhancement of ACE2 binding. These findings inform on novel strategies for therapeutic antibody cocktails.

2022-08-01

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Detection of antigen-antibody affinity by fluorescence autocorrelation spectroscopy In this paper, the specific realization and potential application of fluorescence autocorrelation spectroscopy (FACS) technology as an immunoassay technology were explored. In the experiment, the interaction between Alexa Fluor647 fluorescent molecule and green fluorescent protein antigen and antibody was used as the model, and a desktop fluorescence correlation spectrometer was used to collect the fluorescence autocorrelation data of fluorescent molecular antigen and antibody mixture of different concentrations. Combining the traditional FACS technique with the maximum entropy method, we can quantitatively and qualitatively evaluate the degree of antigen-antibody binding. The acquired fluorescence autocorrelation data were analyzed using self-developed data analysis software to obtain antigen-antibody dissociation constants. The experimental results show that the FACS technology applied to antigen-antibody affinity detection has the characteristics of simplicity, rapidity and sensitivity; the detection limit is between picomolar and nanomolar; the modular data analysis software can standardize the operation process and improve the accuracy of data analysis. and repeatability.

2022-08-01

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