Acta Optica Sinica, Volume. 41, Issue 17, 1730004(2021)
Fluorescence Auto-Correlation Spectroscopy Quantitatively Measures Antigen-Antibody Binding Affinity
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Fig. 2. Flow chart of the data analysis software for determining antigen-antibody binding affinity
Fig. 3. Analyze the affinity of Alex647 fluorescent molecule antigen and antibody using FCS and MEM methods. (a) Normalized fluorescence auto-correlation curves of a standard fluorescent molecule sample, a free Alexa Fluor647 antigen sample, and a bound antigen sample; (b) normalized fluorescence auto-correlation curves of Alexa Fluor647 mixed with different concentrations of monoclonal antibody; (c) distribution curves of diffusion correlation time of the free and bound Alexa Fluor647 obtained by MEM analyses of the 13 antigen-antibody samples; (d) antigen-antibody dissociation constant was obtained by non-linear least squares fitting of the bound antigen fractions data with different concentrations of antibody
Fig. 4. FCS and MEM Analyses of GFP antigen-antibody binding affinity. (a)Normalized fluorescence auto-correlation curves of a standard fluorescent molecule sample, a free GFP antigen sample, and a bound antigen sample; (b) normalized fluorescence auto-correlation curves of samples composed of GFP mixed with different concentrations of monoclonal antibody; (c) distribution curves of diffusion correlation time of the free and bound GFP obtained by MEM analyses of the antigen-antibody samples; (d) antigen-antibody dissociation constant was obtained by non-linear least squares fitting of the bound antigen fractions data with different concentrations of antibody
Table 1. Comparison of FCS with other technologies for quantitatively determining antigen-antibody binding affinity
View tableTable 1. Comparison of FCS with other technologies for quantitatively determining antigen-antibody binding affinity
Technology Theory Characteristics Sensitivity Isothermal Titration Calorimetry (ITC) [ 22 -23 ]By detecting the dynamic heat changes during the reaction process of antibody ligand binding, ITC calculates the binding thermodynamic and kinetic parameters No need to label the sample. Antibody concentration is 15-50 μM. The ligand concentration is 15 times that of the antibody. Sample amount is a few microliters Each test takes 2-2.5 h The small heat change lower than 0.4 μJ in the reaction process cannot be tested The affinity of the antigen and antibody can be detected in the natural binding state Antigen-antibody affinity test with KD value at 10-9-10-2 M Surface Plasmon Resonance (SPR)[ 24 -26 ]When the ligand flows through the antibody fixed on the chip surface at a certain speed and binds to it, SPR calculates the affinity value by fitting the weight changes of the chip during the biding process Sample is fixed on the chip. The sample preparation has to be careful to avoid the damage of the active structure of the sample and produce non-characteristic binding. The detection limit is on the order of 10 pg/mL. Sample amount is micrograms to sub-micrograms Each test takes several minutes Get reaction information of binding kinetics and concentration change during binding Antigen-antibody affinity test with KD value at 10-9-10-3 M Microscale Thermophoresis (MST)[ 22 ]A temperature gradient is induced by an infrared laser. By combining the precise fluorescence detection and sensitive thermophoresis, MST detects the molecular interactions No need to fix the sample, but the sample has to be fluorescent labeled. Sample amount is a few microliters at nanomolar concentration. The ligand concentration is a few mg/mL Each test takes several minutes Energetic detection of affinity. The affinity of the antigen and antibody can be detected in the natural binding state Antigen-antibody affinity test with KD value below 10-9 M Fluorescence Correlation Spectroscopy (FCS)[ 14 ,27 ]By applying the autocorrelation analysis to the fluorescence signal fluctuations during the binding reaction, FCS calculates intermolecular affinity No need to fix the sample, but the sample has to be fluorescent labeled. Sample concentration is pM-nM. Sample amount is tens of microliters Each test takes several seconds The affinity of the antigen and antibody can be detected in the natural binding state Antigen-antibody affinity test with KD value at 10-12-10-7 M
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Yamei Luo, Lin Jian, Haoqi Luo, Keying Huang, Wolun Zhang, Zhulou Wang, Huizhi Zhang, Qian Xiao, Shaohui Huang. Fluorescence Auto-Correlation Spectroscopy Quantitatively Measures Antigen-Antibody Binding Affinity[J]. Acta Optica Sinica, 2021, 41(17): 1730004
Paper Information
Category: Spectroscopy
Received: Jan. 24, 2021
Accepted: Mar. 29, 2021
Published Online: Sep. 3, 2021
The Author Email: Xiao Qian (qianxiao@ibp.ac.cn), Huang Shaohui (shaohuih@ibp.ac.cn)
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