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. 2020 Oct 31;10(11):161.
doi: 10.3390/bios10110161.

A Rapid Immunochromatographic Method Based on a Secondary Antibody-Labelled Magnetic Nanoprobe for the Detection of Hepatitis B preS2 Surface Antigen

Yangyang Cai  1   2   3 , Jun Yan  1   2   3 , Li Zhu  4 , Hengliang Wang  4 , Ying Lu  1   2   3
Affiliations

A Rapid Immunochromatographic Method Based on a Secondary Antibody-Labelled Magnetic Nanoprobe for the Detection of Hepatitis B preS2 Surface Antigen

Yangyang Cai et al. Biosensors (Basel). .

Abstract

Hepatitis B is a globally prevalent viral infectious disease caused by the hepatitis B virus (HBV). In this study, an immunochromatographic assay (ICA) for the rapid detection of hepatitis B preS2 antigen (preS2Ag) was established. The magnetic nanoparticles (MNPs) indirectly labelled with goat anti-mouse (GAM) secondary antibody were applied as a nanoprobe for free preS2 antibody (preS2Ab) capturing and signal amplification. By employing sample pre-incubation processing as well, preS2Ag-preS2Ab was sufficiently caught by the GAM-MNPs probe in 5 min. A qualitative sensitivity of 625 ng/mL was obtained by naked-eye observation within 15-20 min. A standard curve (0-5000 ng/mL) was established, with a quantitative limit of detection (LOD) of 3.6 ng/mL, based on the stability and penetrability of the magnetic signal characteristics. The proposed method for preS2Ag was rapid (~25 min, cf. ELISA ~4 h) and had a good accuracy, which was verified using an ELISA kit (relative error < 15%). Large equipment and skilled technicians were not required. The sensitivity and specificity of the developed GAM-MNPs-ICA method were 93.3% and 90% in clinical serum samples (n = 25), respectively. A good detection consistency (84%) was observed between the developed ICA method and 2 types of commercial ELISA kits, indicating that the GAM-MNPs-ICA has a potential application in large-scale screening for and point-of-care diagnosis of hepatitis B or other infectious diseases.

Keywords: hepatitis B; hepatitis B preS2 antigen; immunochromatographic assay; magnetic nanoparticles; rapid detection.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic illustration of GAM-MNPs-ICA for preS2Ag detection. (a) Preparation of the GAM-MNP probe using the EDC/NHS method. (b) Pre-incubation of preS2Ag, preS2Ab, and GAM-MNPs. (c) Structure of the test strip. (d) Judgment of the negative and positive results and qualitative and quantitative analysis methods.
Figure 2
Figure 2
Characterization of conjugated antigen. (a) UV absorption spectrum. (b) SDS-PAGE. M: Marker; 1: preS2Ag; 2: BSA; 3: preS2Ag-BSA.
Figure 3
Figure 3
Optimization of different T-line concentrations with 0, 50, 500, and 5000 ng/mL of preS2Ag. (a) Qualitative results. (b) Quantitative results. The error bars represented the standard deviation of three repeats (n = 3).
Figure 4
Figure 4
Optimization of the secondary antibody amount coupled onto the surface of MNPs. The error bars represent the standard deviation of three repeats (n = 3).
Figure 5
Figure 5
Qualitative results (a–c) and quantitative results (d) for different concentrations of preS2Ab antibody in sample pre-incubation. The error bars represent the standard deviation of three repeats (n = 3).
Figure 6
Figure 6
Detection sensitivity of the GAM-MNPs-ICA for preS2Ag detection. (a) Qualitative detection results. (b) Quantitative standard curve. The error bars represent the standard deviation of three repeats (n = 3).
Figure 7
Figure 7
Quantitative results of the specificity analysis. The error bars represent the standard deviation of three repeats (n = 3).
Figure 8
Figure 8
Stability evaluation of GAM-MNPs-ICA. The error bars represent the standard deviation of three repeats (n = 3).
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