An epitope or antigenic determinant is the core part of antigen involved in the recognition with an antibody. After antigens, containing numerous epitopes, are recognized by the human immunological system, B lymphocytes will synthesize and secrete miscellaneous antibodies targeting different epitopes to mediate further immunological process. Nowadays, there are many methods that are used to investigate and confirm antigen epitopes, for example, proteolytic cleavage of antigen-monoclonal antibody complexes, proteolytic or chemical cleavage fragment method, Western blotting, PEPSCAN method, chemical modification or mutation analyses, chemosynthesis of peptide, surface display of peptide libraries and random fragment expression libraries, X-ray crystallographic assay and nuclear magnetic resonance spectroscopy assay, and so on . However, most of these methods are complicated, difficult to perform, or of low efficiency. With the development of computer technology and bioinformatics, a set of methods for epitope prediction were developed based on the structural property of amino acid sequences ; the accuracy of prediction of antigenic determinants is about 75% . The methods for epitope prediction can reduce the range of possible epitope and bring us much less workloads for epitope screening. However, it is possible that some of the predicted epitopes exhibit no strong antigenicity. So, developing a novel method to analyze the antigenicity of predicted peptides has become an urgent requirement for epitope determination.
Fluorescence polarization (FP) is a unique and powerful technique for the rapid analysis of interactions of small molecular weight molecules (labeled with fluorophore) and macromolecules. The theory of fluorescence polarization was for the first time described in 1926 by Perrin . When fluorescent molecules in solution are excited by a plane-polarized light beam with an appropriate wavelength, they emit fluorescent signals back into the same polarized plane, provided that the molecules remain stationary. However, if the excited molecules rotate or tumble while in the excited state, then fluorescence is emitted into a plane different from the plane used for excitation. Therefore, if the viscosity and temperature of a solution are kept constant, the degree of fluorescence polarization is dependent on the molecular volume, that is, the size of a fluorescent molecule. FP assay is based on the rotational differences between a small soluble molecule in solution (labeled with a fluorochrome) and the small molecule combined with its ligand. A small molecule can rotate randomly at a rapid rate, resulting in rapid depolarization of light, while a larger complex molecule can rotate slower and depolarize light at a reduced rate. The rate change in depolarization can be measured. High polarization values indicate that the small molecule is reacting with its ligand or target molecule, and low values mean that there is no small molecule ligand or small molecule to react with target molecule. Nowadays, homogeneous FP assays have been successfully applied to many research fields, including DNA-protein, protein-protein, and antigen-antibody binding [5–11]. However, the current FP assay is based on organic dye labeling, having some problems such as intrinsic photobleaching and low-emission efficiency, and how to solve these questions has become a great challenge.
Quantum dots have been subject to intensive investigations due to their unique properties and potential application prospect [12–14]. So far, several methods have been developed to synthesize water-soluble quantum dots (QDs) for use in biologically relevant studies [15–18]. QDs exhibit high quantum yield, high photostability, and size-dependent tunable emission, being attractive alternative luminescent labels for ultrasensitive detection and molecular imaging. For example, QDs have been used successfully in cellular imaging [19, 20], immunoassays , DNA hybridization , and optical barcoding . Quantum dots provide a new functional platform for bioanalytical sciences and biomedical engineering. Therefore, it is feasible to use QD labeling to improve the FP technique for detection of tumor biomarkers in patient sera [24, 25].
If micromolecular antigens are adopted, FP assays can also be used to analyze the interaction of the antigen and its antibody. Herein, we reported a CdTe quantum dot-based method to screen rapidly antigenic epitopes. All possible antigenic epitopes from hepatitis B virus (HBV) surface antigen protein were predicted, and the antigenicity of peptide was determined by analyzing the recognition and combination of peptide and standard antibody samples using the FP technique. Subsequently, the immunodominant epitopes of HBV surface antigen in Chinese people with positive anti-HBV surface antigen were screened using the same method. Besides, the application of the obtained dominant antigenic peptides in detecting anti-HBV surface antibody was also investigated by FP assay.