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Immune Adherence Hemagglutination Assay (IAHA)

After the successful application of IAHA tests to the measurement of some animal viruses (herpes simplex virus, simian virus 40, adeno- and enteroviruses) and their antibodies in [76], this method has been investigated and applied in the detection of other viruses as well as antibody responses to them. For example, an IAHA test conducted by Kapikian et al. [77] was applied for the detection of antibody against Norwalk virus from acute epidemic nonbacterial gastroenteritis, by using purified viruses from stool as an antigen. Around the late s, IAHA tests began to replace EM and IEM for the detection of NoVs in clinical samples [78]. IAHA used the agglutination of human erythrocytes with the antigen-antibody complex to study HuNoV sero-prevalence. Though this method is rapid, simple, and inexpensive, its inability to differentiate immunoglobulin subclasses as well as the problem of the virus agglutinating red blood cells inhibits its application. In addition, the method is not suitable for direct NoV detection in fecal samples [79,80].

Radio-Immunoassays (RIA)

Ultimately, IAHA was replaced by RIA, which used radio-labelled immunoglobulin G (IgG) to detect NoV antigen or antibody. RIA is often conducted in a microtiter format and could be used to detect viral antigens (or antibodies) by involving a competitive binding between unlabeled and radioisotope (I), labeled corresponding antibodies (or antigens). In , a study by Greenberg et al. [81] using a microtiter solid-phase RIA successfully detected Norwalk virus (from stool samples) and its antibody with a much higher sensitivity than IAHA assay. When utilized for the detection of NoV antigen in stool samples, the IgG which was purified from a convalescent serum of a NoV infected patient was used as an antibody and radiolabeled. The radiolabeled IgG was then added to competitively bind with NoV antigen against unlabeled IgG. A reduction of 50% or more radioactivity defines the presence of the viral antigen.

The blocking RIA assay for NoV antibody detection was 10 to folds more sensitive than IAHA and required much less antigen [82]. There have been a number of studies however, where the RIA assay was not able to detect any NoV strains [83]. Since both IAHA and RIA methods require reagents from human volunteers, their applications for the detection of HuNoV are considered to be limited [82].

Enzyme Immunoassays (EIA)

Enzyme immunoassays (EIA) incorporate specific viral antibodies (or antigens) for the detection of viral antigens (or antibodies). EIA is similar to RIA, but utilizes enzymes rather than radioisotopes for detection. Aside from not having the potential of being exposed to radioisotopes, EIA has several advantages when compared to RIA, which includes the increased stability of reagents used, decreased assay time, and simpler equipment operation. Specifically, the radioisotope labelled antibody only has several days to weeks of shelf-life, while the antibody labelled by the enzyme or biotin has a shelf-life of several months. In addition, the assay time can be reduced by several days when compared to RIA [82]. The use of colorimetric and bioluminescent EIA methods for HuNoV detection will be discussed here in detail.

In the s, the cloning of NoV capsid protein enabled the production of NoV VLPs. These were produced from baculovirus recombinants that contained NoV VP1. In particular, NoV VLPs were shown to be antigenically and morphologically similar to native NoVs, and thus have been widely used for the structural studies of NoV [84]. The use of VLPs has also allowed the detection of viral antibodies [85].

Cloned NoV capsid protein can also be used to induce antibodies against NoVs, which can then be applied in enzyme-linked immunosorbent assay (ELISA) for detection. A number of commercial ELISA kits are available for NoV detection (both GI and GII). The two most commonly used kits are IDEIA Norovirus (Oxoid Ltd., Hampshire, United Kingdom; two generations available) and RIDASCREEN Norovirus (R-Biopharm, Darmstadt, Germany; three generations available). There are wide ranges of reported sensitivity and specificity for these kits. The sensitivities for IDEIA and RIDASCREEN Norovirus range from % to % and % to %, respectively, while their specificities range from % to % and % to %, respectively [86]. Several factors contribute to the different performance of commercial EIA kits in terms of detection of norovirus outbreaks. These include the viral titer and viral genotypes present in clinical samples (since the antibodies in EIA kits have varied affinities to different NoV genotypes), specimen collection (the time collected after symptoms onset), infection extent (outbreak or sporadic), and patient demographics (pediatric or adults) [86].

Compared to the 2nd generation IDEIA ELISA test, the 3rd generation RIDASCREEN ELISA has much higher sensitivity for the detection of NoVs. According to a comparison study of these two kits by Kirby et al. [87], the sensitivity of the 3rd generation RIDASCREEN was 63% and the specificity was more than 98%, while the sensitivity of IDEIA was only 45%. Due to its combination of several cross-reactive monoclonal and polyclonal antibodies, RIDASCREEN has the capability to detect some HuNoV genotypes. Despite these advantages, RIDASCREEN tests generally have low sensitivity. In particular, samples from sporadic NoV cases with GI and mixed infections (GI/GII) were unlikely to be detected by the kit [88]. With a LOD of 10⁴&#x;10⁶ viral particles/mL of stool by ELISA, it is not likely to be sensitive enough for detection of NoVs in food or environmental samples [78]. Moreover, since HuNoVs exhibit at least dissimilar antigenic behaviors, the development of an EIA that is cross-reactive to all HuNoV genotypes is difficult [11].

Considered to be more than just a routine ELISA test, a bioluminescent enzyme immunoassay (BLEIA) conducted by Sakamaki et al. [89], was reported to be able to detect HuNoV VLPs, including 6 GI and 8 GII. This assay had a good reproducibility, with a turnaround time of 46 min and a throughput of tests/h. A similar BLEIA method by Shigemoto et al. [90] could detect 3 NoV GI genotypes and 10 GII genotypes from fecal samples with a LOD of 10⁶ gc per gram of stool and below and a sensitivity of %. Similar results were observed in the study by Suzuki et al. [91], with higher sensitivity (%), specificity (%) and detection rate (%) of BLEIA method compared to the reverse transcription loop-mediated isothermal amplification (RT-LAMP) method, for the detection of HuNoVs from fecal samples. It was found in these studies that BLEIA assays did not show cross-reactivity towards bacteria or other enteric viruses and the sensitivity was around 10⁵&#x;10⁶ copies/g stool samples, which is roughly comparable to that observed for ELISA.

Immuno-Chromatography (ICG)

Aside from the use of commercial ELISA tests, a number of commercial ICG lateral flow assay (LFA) kits are also available for NoV detection (both GI and GII). ICG kits have a number of advantages, including a short timeline (result achieved within 15~30 min), long shelf life (12~24 months), ease of use, relatively low cost (not requiring specific laboratory equipment), and relative ease in manufacturing. Such kits include Ridaquick Norovirus (R-Biopharm, Darmstadt, Germany), SD Bioline Norovirus (Standard Diagnostics, Inc., Kyonggi-do, South Korea), ImmunoCardSTAT!^® Norovirus (Meridian Bioscience Europe, Nice, France), and NOROTOP^® (manicapital.com SA, Strasbourg, France). A study comparing these four kits concluded that the clinical sensitivity for the detection of GII.4 stool samples is 78%, 59%, 61%, and 67%, respectively [92]. The ICG kits, including two commonly used kits (Ridaquick and SD Bioline Norovirus), are shown to have a wide variability of performance, due to similar reasons as those described for ELISA kits, and are related to challenges with the antibodies used. Specifically, the sensitivities for Ridaquick and SD Bioline Norovirus range from % to % and % to %, respectively, while the specificities for Ridaquick and SD Bioline Norovirus range from % to % and % to %, respectively [86]. Due to the wide variation of the sensitivity and specificity, more sensitive molecular detection methods are recommended for samples that have negative results from the ELISA or ICG kits [86].

The detection of the Ridaquick Norovirus ICG kit is based on incorporating gold-labeled anti-NoV antibodies and biotinylated anti-NoV antibodies. A streptavidin test line could capture the sandwich complex, while the unbound complex would migrate to the control line. The high LOD of traditional LFA by using blue latex or AuNPs could be improved by pre-concentration or the use of other reporter particles (e.g., enzyme labeled particles, photoluminescent particles, and phage nanoparticles). An example is the LFA study conducted by Hagström et al. [93], who incorporated M13 phage nanoparticles as reporters, and an antibody pair to detect GI.1 Norwalk VLPs. This assay gave a LOD of 10⁷ VLPs/mL, which was fold more sensitive than a conventional gold nanoparticle LFA test.

Western Blot

The western blot (also called protein immunoblot) assay has been widely used for the analysis of proteins in tissue homogenates or extracts and its use is composed of several steps. First, the proteins of the analytes are separated by sodium dodecyl sulfate&#x;polyacrylamide gel electrophoresis (SDS-PAGE), according to their molecular weight. Then the protein bands are transferred to a nitrocellulose membrane, followed by blocking and the addition of a specific primary antibody and enzyme labeled secondary antibody. The step of gel electrophoresis reduces the probability of the cross-reactivity of antibodies. Therefore, western blot seldom gives false positive results and it is the most commonly used technique to confirm the positive results from ELISA tests [94]. However, compared to ELISA tests, western blot is more difficult to perform and it requires higher skill.

Western blot has been used for the detection of NoV GII.4 VLPs. The VLPs are separated by SDS-PAGE, then IgG and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG were incorporated to capture and detect VLPs. The immunogenicities of VLPs and P particles were studied by western blot, and the results showed that VLPs had higher immunogenicity than P particles [95].

Western blot has also been used in the study of mapping NoV antibodies. By testing the interaction of mAbs with the deletion mutants of GII.4 VLP, the binding sites of the antibodies could be identified [55]. Finally, Western blot has also been applied in the evaluation of antibody response after viral infection, by using a crude small round-structured virus sample as an antigen. The major viral structural protein could also be determined by Western blot [96].