# Advances and Future Perspective on Detection Technology of Human Norovirus
## 2 Detection Techniques
### Immunological Methods  
Early immunological methods were based on the specific reaction between patients' serum and HuNoV particles, for example, immune-adhesive haemagglutinin assay (IAHA), radioimmunoassay (RIA), biotin-avidin immunoassay (BAI) and enzyme immunoassay (EIA) [30; 31; 32; 33]. The IAHA can be used to assess the level of Norwalk virus antibodies in serum, but it cannot detect viral particles in clinical samples. The RIA used radioisotopes (I\({}^{125}\)) to label antibodies (anti-NoV IgG). However, the shelf life of labelled  
Figure 1: Structural and nonstructural proteins of Human norovirus. antibodies was short (less than 2 weeks), and the detection was time-consuming (approximately 6 days). It also had disadvantages such as high cost and a certain degree of risk. Compared with RIA, the shelf lives of labelled antibodies for BAI and EIA were longer, which was 3 months (\(-20\)\({}^{\circ}\)C) and 6 months (4 \({}^{\circ}\)C), respectively. In brief, these methods primarily relied on a limited number of serum and clinical stool samples from infected volunteers. Moreover, the major shortcomings, such as long processing time, difficult matching of serum and limited detection sensitivity for clinical samples, hindered the development of immunological detection technology. The Norwalk virus genome was successfully cloned until 1992. The VP1 was further expressed by the baculovirus system. The VP1 could self-assemble to form virus-like particles, which had the same immunogenicity as NoV. It provided an excellent antigen and antibody production platform and served as an alternative model to study the interaction between receptors from potential host cells and NoVs [34; 35]. Since then, monoclonal antibodies (MAbs) and polyclonal antibodies (PAbs) against VLPs have been produced and widely used in the immunological detection of HuNoVs. Studies showed that hyperimmune serum obtained from VLP-immunised animals (mice, guinea pigs and rabbits) contributed high specificity and sensitivity to RIA, EIA and enzyme-linked immunosorbent assay (ELISA) detection methods compared with human serum samples [34; 36]. In 1995, [PERSON] et al. prepared MAbs against the NoV prototype 8 FIIa strain. It was applied in EIA to detect NoV in stool samples [37]. The LOD was 1 ng/mL. The specificity was strong, and cross reaction was not detected. In 2007, [PERSON] et al. used anti-GII3 VLP PAbs as capture antibodies and three anti-GII4 VLP MAbs as detection antibodies to conduct a sandwich ELISA. A total of eight genotypes (GI and GII) of HuNoVs from clinical samples were successfully detected [38]. In 2008, [PERSON] et al. developed an immunomodotropatic (ICG) test strip for the rapid detection of GI and GII HuNoVs in clinical stool samples [39]. [PERSON] et al. developed a colloidal gold ICG assay for the detection of HuNoVs [40]. This system used the recombinant S protein of HuNoVs as the immunogen to prepare MAbs (capture antibodies and labelled antibodies) for the rapid (15 min) detection of GI and GII in clinical samples. Compared with RT-qPCR, the detection rate, specificity and consistency were 84.2%, 100.0% and 87.7%, respectively. A total of 10 genotypes of HuNoVs could be detected simultaneously. In addition, alkaline lysis was performed on HuNoV particles during pretreatment. This process could obtain several viral particles, and it is safe for people. Moreover, this method was easy to operate without the extraction of viral nucleic acids. Therefore, it is suitable for use as a point-of-care test or for preliminary screening of HuNoVs. Commercial kits for the detection of HuNoVs based on immunological methods were developed. EIA-based kits mainly include IDEIA Norovirus (Oxoid Ltd., Hampshire, United Kingdom; two generations available) and RIDASCREEN Norovirus (R-Biopharm, Darmstadt, Germany; three generations available).