Abstract:

Early diagnosis of Epstein-Barr virus (EBV) can reduce the risk of major illnesses. Disadvantages of EBV antibody detection methods that are commonly used clinically include lengthy assay time, need for a lot of reagent, and low efficiency. Compared with traditional detection methods, microfluidics technology offers high throughput, low reagent consumption, less bio-contamination, and a higher degree of automation. Advantages of magnetic immunofluorescence technology include high detection efficiency and a strong signal. The combined advantages of the two methods can compensate for the shortcomings of traditional methods. In the present study, polymethyl methacrylate (PMMA) as the raw material was subjected to laser cutting and vacuum hot pressing to quickly obtain chips. Magnetic beads labeled with antigen and fluorescent microspheres labeled with anti-human antibody were then rapidly lyophilized into microspheres by freeze-drying and embedded into the chips. After incubation and cleaning, the last step was detection. Image J software was used to analyze the mean fluorescence intensity and obtain negative or positive test results. To determine the precision of the chip, high- and low-value samples of each item were retested 10 times. The mean values were calculated to obtain the relative standard deviation (RSD) for several common pathogens. Furthermore, the coincidence rate of clinical samples was tested using a chemiluminescence immunoassay (CLIA) to determine the potential clinical application value. The RSD of the precision test for each item was <10%, indicating good precision. The precision of the accelerated stability test was not verified. Specificity test results revealed no cross-reaction with some common pathogen antibodies, indicating good specificity. It remains to be verified whether the antibodies detected by this method cross-react with other herpes simplex viruses, such as types 1 and 2, Kaposi’s sarcoma-associated virus, and human herpes virus type 6 and 7. Of the 121 clinical samples tested, statistical analysis of the data indicated good agreement with the chemiluminescence immunoassay in clinical trials. EB viral capsid antigen (EB VCA) IgG positive coincidence rate was 95.77% (68/71), the negative coincidence rate was 86% (43/50) (Kappa=0.828, P<0.05), the limit of detection (LOD) was 1.92 U/mL, and the linear range was 1.92 to 200 U/mL. The EB VCA IgA positive coincidence rate was 92% (46/50), negative coincidence rate was 92.96% (66/71) (Kappa=0.847, P<0.05), LOD was 2.79 U/mL, and the linear range was 2.79 to 200 U/mL. The positive coincidence rate of EB nuclear antigen 1 (EB NA1) IgG was 92.96% (66/71), the negative coincidence rate was 92% (46/50) (Kappa=0.847, P<0.05), the LOD was 3.13 U/mL, and the linear range was 3.13 to 200 U/mL. The positive coincidence rate of EB NA1 IgA was 90% (45/50), the negative coincidence rate was 91.55% (65/71) (Kappa=0.813, P<0.05), the LOD was 1.53 U/mL, and the linear range was 1.53 to 200 U/mL. Compared with the traditional enzyme-linked immunosorbent assay, the novel method featured a shorter detection time, reduced use of reagent, high degree of automation, and less bio-contamination. Compared with CLIA, advantages of the novel method include multi-item combined detection, long luminescence time, and simple use as a basic health service. Compared with silicon and ceramic microfluidic chips, advantages of the selected PMMA material include low processing cost, short processing time, simple processing technology, and easy industrialization. A refinement that can still be made include the use of molding instead of laser cutting technology, which can further shorten the chip processing time. In summary, a microfluidic detection platform was initially built to provide a rapid, sensitive, simple, highly automated, and easy to be used by basic health service for the quantitative combined detection of EBV VCA and EB NA1 IgG and IgA.

Key words: microfluidic, magnetic immunofluorescence, Epstein-Barr virus (EBV), rapid detection