Comparison of six antibody assays and two combination assays for COVID-19.

INTRODUCTION: In this work, six SARS-CoV-2-specific antibody assays were evaluated, namely, two pan-immunoglobulin (pan-Ig) assays [Roche Elecsys Anti-SARS-CoV-2 (named "Elecsys" in this study) and the PerkinElmer SuperFlex™ Anti-SARS-CoV-2 Ab Assay (SuperFlex_Ab)], two IgM assays [SuperFlex™ Anti-SARS-CoV-2 IgM Assay (SuperFlex_IgM) and YHLO iFlash-SARS-CoV-2 IgM (iFlash_IgM)], and two IgG assays [SuperFlex™ Anti-SARS-CoV-2 IgG Assay (SuperFlex_IgG) and iFlash-SARS-CoV-2 IgG (iFlash_IgG)]. Combination assays of SuperFlex™ (SuperFlex_any) and iFlash (iFlash_any) were also evaluated. METHODS: A total of 438 residual serum samples from 54 COVID-19 patients in the COVID-19 group and 100 samples from individuals without evidence of SARS-CoV-2 infection in the negative control group were evaluated. RESULTS: In the early stage of COVID-19 infection, within 14 days of symptom onset, the seropositive rate was lower than that of the late stage 15 days after onset (65.4% vs 99.6%). In the total period, the pan-Ig and IgG assays had higher sensitivity (90.8-95.3%) than the IgM assays (36.5-40.7%). SuperFlex_Ab and SuperFlex_any had higher sensitivity than Elecsys and SuperFlex_IgG (p < 0.05). The specificity of all the assays was 100%, except for SuperFlex_IgM (99.0%). The concordance rate between each assay was higher (96.4-100%) in the late stage than in the early stage (77.4-98.1%). CONCLUSION: For the purpose of COVID-19 diagnosis, antibody testing should be performed 15 days after onset. For the purpose of epidemiological surveillance, highly sensitive assays should be used as much as possible, such as SuperFlex_Ab, iFlash_IgG and their combination. IgM assays were not suitable for these purposes.

Non-structural protein 1 (NS1) antibody-based assays to differentiate West Nile (WN) virus from Japanese encephalitis virus infections in horses: effects of WN virus NS1 antibodies induced by inactivated WN vaccine.

Antibodies to non-structural protein 1 (NS1) of West Nile virus (WNV) have been used to differentiate WNV infection from infection by serologically cross-reactive flaviviruses, including Japanese encephalitis virus (JEV), in horses. However, since the inactivated West Nile (WN) vaccine has been reported to induce NS1 antibodies, there is concern about the reliability of using NS1-based assays for testing vaccinated horses. Therefore, the effect of inactivated WN vaccine-induced antibodies on an epitope-blocking ELISA and complement-dependent cytotoxicity (CDC) assay were investigated. Both assays are based on NS1 antibodies and were established previously to differentiate WNV from JEV infections in horses. Groups of three horses were vaccinated with two or three doses of a commercial inactivated WN vaccine and NS1 antibodies were detected by a conventional ELISA after the second vaccination. Vaccine-induced NS1 antibodies were also detected by blocking ELISA and a CDC assay and affected the ability of these assays to differentiate WNV from JEV infections. However, the effect was less significant in the CDC assay, where use of a low serum concentration ensured effective differentiation. The more efficient detection of infection-induced antibodies over vaccine-induced antibodies by the CDC assay was potentially attributable to the different IgG isotype profiles of these antibodies.

Specific antibody responses to West Nile virus infections in horses preimmunized with inactivated Japanese encephalitis vaccine: evaluation of blocking enzyme-linked immunosorbent assay and complement-dependent cytotoxicity assay.

West Nile virus (WNV) and Japanese encephalitis (JE) virus are distributed separately in the world with some exceptions. There is a concern that WNV may invade into Asia where JE virus exists. On and after such invasion, any differential diagnosis could be complicated by serological crossreactivities. We previously demonstrated experimentally using horses infected with WNV that preimmunization with inactivated JE vaccine considerably affected the ability of neutralization tests and immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (ELISA) to diagnose WNV infection. Here, we investigated WNV-specific antibody responses in vaccinated horses using a blocking ELISA and complement-dependent cytotoxicity (CDC) assay to evaluate these two newly developed serodiagnostic methods for WNV infection. Sera previously collected from six experimentally infected horses were used: Three were vaccinated before the infection, whereas the other three remained unvaccinated. WNV-specific antibody responses were successfully detected in the vaccinated and unvaccinated horses using both new methods, except for one vaccinated horse in which responses were not induced, probably as a result of crossprotection induced by JE vaccination. Specific antibody responses were at earliest detected from days 9 to 10 postinfection in the blocking ELISA, whereas the CDC assay provided earlier detection (at days 7-8) in all horses. The time courses of antibody levels were similar between vaccinated and unvaccinated horses in either method, indicating no notable effect of vaccination on detection of specific antibody responses, as far as antibodies were induced. These results indicated that blocking ELISA, but preferably the CDC assay, can be useful for detecting WNV infection in JE-vaccinated horses.

Complement-dependent cytotoxicity assay for differentiating West Nile virus from Japanese encephalitis virus infections in horses.

A complement-dependent cytotoxicity (CDC) assay was established to measure antibodies to the West Nile virus (WNV) nonstructural protein 1 (NS1) in horses. Sera collected from a WNV-infected horse mediated lysis of WNV NS1-expressing cells in a dose-dependent manner at higher percentages than sera from a Japanese encephalitis virus (JEV)-infected horse. The percentages of specific lysis for sera diluted 1:10 to 1:80 were <19.8% (assay cutoff) for almost all of the 100 JEV-infected or uninfected horses tested, in contrast to 55 to 76% in WNV-infected horses. Experimental infection revealed that horses became anti-WNV NS1 antibody positive 10 days after WNV infection. This study demonstrated the utility of this assay for differentiating WNV from JEV infections in horses.

Antibodies to bovine serum albumin in human sera: problems and solutions with casein-based ELISA in the detection of natural Japanese encephalitis virus infections.

An ELISA system for measuring antibodies to nonstructural protein 1 (NS1) of Japanese encephalitis virus has already been established. This system uses an ELISA diluent containing casein, instead of bovine serum albumin (BSA). During a survey, we found that 21 (21%) of 102 children aged 1-5 years, who had no history of Japanese encephalitis vaccination and were without detectable neutralizing antibodies, showed positive results with this ELISA system. Western blotting analysis showed that sera from 19 (91%) of these 21 subjects had antibodies to BSA, but not NS1. These sera reacted with BSA antigen remaining in immunoaffinity-purified NS1 antigen. One solution to this problem was to reduce the BSA level to < or =1% of the NS1 amount. Another was to use a control well sensitized with BSA with the same amount as that contained in the NS1 antigen preparation.

Natural Japanese encephalitis virus infection among humans in west and east Japan shows the need to continue a vaccination program.

Japanese encephalitis (JE) is a serious disease in Asia, but it can be prevented by vaccination. To evaluate the necessity for vaccination in areas with reduced numbers of vector mosquitoes, as well as patients, it is critical to understand the frequency of natural virus exposure. An antibody survey was recently conducted to estimate current natural infection rates in Japan, where the vaccination rate has dropped in recent years. Serum samples were collected in 2004-2008 from inhabitants of Kumamoto Prefecture in west Japan, and in 2004-2006 from the Tokyo Metropolitan area of east Japan. Average annual infection rates estimated from the prevalence of antibodies to the nonstructural 1 protein (NS1) of JE virus was 1.8% in Kumamoto and 1.3% in Tokyo. When estimated from percentages of populations with detectable neutralizing antibodies but with no vaccination history, the average annual infection rate was 2.6% in both survey areas. Thus, JE virus remains present and active in nature in Japan. Therefore, continuing a vaccination program is indispensable to prevent JE infection in humans.

Detection by ELISA of antibodies to Japanese encephalitis virus nonstructural 1 protein induced in subclinically infected humans.

Japanese encephalitis (JE) is a fatal mosquito-borne disease that is vaccine-preventable. The natural infection rate is a critical factor for evaluations of the necessity for vaccination. Detection of antibodies to virus nonstructural (NS) proteins is a theoretical strategy to survey natural infections among populations vaccinated with an inactivated JE vaccine consisting of only structural proteins. Here, we present our development of an enzyme-linked immunosorbent assay (ELISA) to detect low levels of NS1 antibodies induced in humans with subclinical infections. We used a casein-based ELISA diluent to minimize nonspecific reactions. A tentative cut-off value (0.185) was statistically calculated from NS1 antibody levels obtained with healthy American individuals negative for antibodies to JE virus. Comparison with our previously developed immunostaining method provided a significant correlation coefficient (0.764; P<0.001) and high qualitative agreement (82.5%). The presence of NS1 antibodies in sera was confirmed by Western blotting analysis. Using serially collected sera, we estimated the duration of NS1 antibodies between seroconversion and seroreversion to be 4.2 years.

Prevalence of antibodies to Japanese encephalitis virus among inhabitants in Java Island, Indonesia, with a small pig population.

Japanese encephalitis virus (JEV) is maintained through a transmission cycle between amplifier swine and vector mosquitoes in a peridomestic environment. Thus, studies on natural JEV activities in an environment with a small size of pig population have been limited. Here, we surveyed antibodies against JEV in inhabitants of Jakarta and Surabaya located in Java Island (Indonesia), which has a small swine population. Overall, 2.2% of 1,211 sera collected in Jakarta and 1.8% of 1,751 sera collected in Surabaya had neutralizing antibody titers of >or= 1:160 (90% plaque reduction). All the samples with titers of >or= 1:160 against JEV were also examined for neutralizing antibodies against each of four dengue viruses to confirm that JEV antibody prevalences obtained in the present survey were not attributable to serologic cross-reactivities among flaviviruses distributed in Java. These results indicated that people in Java Island are exposed to natural JEV infections despite a small swine population.

Utilization of complement-dependent cytotoxicity to measure low levels of antibodies: application to nonstructural protein 1 in a model of Japanese encephalitis virus.

Enzyme-linked immunosorbent assay (ELISA) and related assays are representative of methods currently used for antibody tests. However, they occasionally produce nonspecific reactions, thus making it difficult to reliably measure low levels of specific antibodies. To find a test method that minimizes nonspecific reactions, we introduced the principle of antibody-mediated complement-dependent cytotoxicity (CDC) into an antibody assay. The procedure has three steps: (i) the mixing of test samples with a suspension of cells expressing the antigen of interest on their surfaces, (ii) the addition of rabbit complement, and (iii) the measurement of lactose dehydrogenase (LDH) activities by adding a chromogenic substrate to the reaction mixture. When the specific antibodies exist in the sample, complement activation triggered by antibody binding on the surface of the antigen-expressing cells may lyse the cells, releasing LDH into the medium. Mouse and rabbit sera hyperimmune to nonstructural protein 1 (NS1) of Japanese encephalitis virus (JEV) lysed NS1-expressing cells in a dose-dependent manner. Evaluations using sera from horses naturally infected with JEV showed that the CDC assay had quantitative correlation and qualitative agreement with previously established NS1 antibody-detecting immunostaining and ELISA methods. The assay method also detected NS1 antibodies in sera of mice 2 days after experimental infection with JEV; specific, but not natural, immunoglobulin M antibodies were detected. Since almost all sera examined in this study showed no nonspecific reactions, the CDC assay was shown to be a reliable method for measuring low levels of specific antibodies.

Epitope-blocking enzyme-linked immunosorbent assay to differentiate west nile virus from Japanese encephalitis virus infections in equine sera.

West Nile virus (WNV) is now widely distributed worldwide, except in most areas of Asia where Japanese encephalitis virus (JEV) is distributed. Considering the movement and migration of reservoir birds, there is concern that WNV may be introduced in Asian countries. Although manuals and guidelines for serological tests have been created in Japan in preparedness for the introduction of WNV, differential diagnosis between WNV and JEV may be complicated by antigenic cross-reactivities between these flaviviruses. Here, we generated a monoclonal antibody specific for the nonstructural protein 1 (NS1) of WNV and established an epitope-blocking enzyme-linked immunosorbent assay that can differentiate WNV from JEV infections in horse sera. Under conditions well suited for our assay system, samples collected from 95 horses in Japan (regarded as negative for WNV antibodies), including those collected from horses naturally infected with JEV, showed a mean inhibition value of 8.2% and a standard deviation (SD) of 6.5%. However, inhibition values obtained with serum used as a positive control (obtained after 28 days from a horse experimentally infected with WNV) in nine separate experiments showed a mean of 54.4% and an SD of 7.1%. We tentatively determined 27.6% (mean + 3 x SD obtained with 95 negative samples) as the cutoff value to differentiate positive from negative samples. Under this criterion, two horses experimentally infected with WNV were diagnosed as positive at 12 and 14 days, respectively, after infection.