Quantitative detection of antibody based on single-molecule counting by total internal reflection fluorescence microscopy with quantum dot labeling.
We presented a sensitive method to quantify antibody based on single-molecule counting by total internal reflection fluorescence microscopy with quantum dot labeling. In this method, the biotinylated monoclonal anti-human IgG molecules were immobilized on the silanized glass substrate surface. By the strong biotin-streptavidin affinity, streptavidin-coated quantum dots were labeled to the target molecules as fluorescent probe. Then, images of fluorescent spots in the evanescent wave field were obtained by a high-sensitivity electron multiplying charge-coupled device. Finally, the number of fluorescent spots corresponding to single molecules in the subframe images was counted, one by one. The linear range of 8.0 x 10(-14) to 5.0 x 10(-12)molL(-1) was obtained between the number of single molecules and the sample concentration.
Jiang
Dafeng
D
School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, PR China.
Wang
Lei
L
Jiang
Wei
W
D000906:Antibodies / Q000032:analysis*; D000888:Antibodies, Anti-Idiotypic / Q000032:analysis; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D045663:Quantum Dots; D012680:Sensitivity and Specificity
D000906:Antibodies; D000888:Antibodies, Anti-Idiotypic; C048844:anti-IgG