The six assays and two combination assays for COVID-19 antibody testing were compared using 438 serum samples from 54 confirmed COVID-19 patients and 100 samples from COVID-19-negative individuals in this study. The 2 pan-Ig assays (Elecsys and SuperFlex_Ab), two IgG assays (SuperFlex_IgG and iFlash_IgG), and two combination assays (SuperFlex_any and iFlash_any) had high sensitivities, specificities, PPVs and NPVs. Moreover, the positivity rates of these assays among COVID-19-confirmed patients were also high, especially 15 days after symptom onset. On the other hand, the positivity rates and sensitivities of the IgM assays (SuperFlex_IgM and iFlash_IgM) were lower than those of the pan-Ig assays, IgG assays, and combination assays in each period. At least in this study, there was no evidence that the IgM assays were superior to the pan-Ig or IgG assays. Therefore, pan-Ig assays, IgG assays and combination assays are all considered appropriate for both epidemiological studies and clinical use.
Although the usefulness of antibody testing for SARS-CoV-2 remains controversial, the CDC suggests its importance for public health and clinical use, e.g., for monitoring and responding to the COVID-19 pandemic . They recommend choosing an assay with very high sensitivity and specificity. PerkinElmer SuperFlex™ contains 3 assays, pan-Ig, IgG, and IgM assays, and the pan-Ig and IgG assays have high sensitivities and remarkably high specificities. SuperFlex™ can also be used with the orthogonal testing algorithm. Therefore, SuperFlex™ is a feasible way to adhere to the CDC's recommendation.
Fifteen days after onset, pan-Ig assays, IgG assays and combination assays were positive, with a high positivity rate. On the other hand, in the early clinical stage, the positivity rate was low, and the concordance rate varied depending on the assay. These tendencies have also been observed in former studies [11,12,13]. In particular, SuperFlex_Ab and SuperFlex_any had significantly higher sensitivities than Elecsys—one of the FDA EUA assays. SuperFlex_Ab, SuperFlex_any, iFlash_IgG, and iFlash_any demonstrated the same performance in detecting SARS-CoV-2-specific antibodies and had relatively high sensitivity, even in the early stage. Increased sensitivity in the early stage is a great benefit for clinical diagnosis and serosurveillance. Although pan-Ig assays and IgG assays had good sensitivities relative to other than IgM assays, their performance in the early stage was suboptimal.
There remains no gold standard for COVID-19 antibody testing. One feasible strategy is to evaluate antibody assays using neutralizing antibodies as the standard. Of course, for the evaluation of convalescent plasma and vaccination, neutralizing assays should be used as the standard. However, for SARS-CoV-2-specific antibody testing assays, as used in this study, the sensitivity of antibody detection could be higher than that for neutralizing antibody testing for diagnosis. Moreover, the procedures are more complex for neutralizing assays than for antibody testing. Therefore, from a standpoint of seroprevalence, SARS-CoV-2-specific antibodies instead of neutralizing antibodies could be more easily detected and appropriate for study. Therefore, we defined seropositive as an antibody-positive result obtained from at least one assay among seroconversion-observed patients. However, this strategy may cause false-positive results. Each assay is a semiquantitative assay, and the antibody titer can increase. Even if only one positive result was obtained from one patient, the other assays could reveal a lower antibody titer increase that is below the cutoff level. In the early stage, between day 1 and day 14 after symptom onset, SuperFlex_Ab, SuperFlex_any, and iFlash_IgG had approximately 80% sensitivity. As described in a previous study, it is difficult to determine the optimal assay for the detection of SARS-CoV-2-specific antibodies during this period [11,12,13]. Fifteen days after symptom onset, the pan-Ig, IgG assays, and combination assays had remarkably high sensitivities, and no significant difference was found among them.
This study has several limitations, which should be acknowledged. First, the COG in this study included mainly COVID-19 patients hospitalized with severe illness and a small number of mild or asymptomatic patients. Some studies described negative results for antibody testing among patients who were asymptomatic or who had mild disease . The patient characteristics widely differed, and the timing of blood sample collection could not be matched. Second, this study population included a relatively small number of patients at 56 days after onset. Further evaluation is needed to understand how long antibodies can be detected among COVID-19 patients. Third, a neutralizing antibody assay could not be performed. Therefore, it remains uncertain whether the antibody tests analyzed in this study can be correlated with that of neutralizing assays. Finally, the SARS-CoV-2 antibody assay that targets the spike protein cannot distinguish between antibodies derived from infection and vaccination. However, it remains valid for testing individuals who have not been vaccinated and for screening use for SARS-CoV-2 antibody detection. For instance, the SuperFlex™ assay can be performed easily and has a short turn-around time (15 min). Among the post-vaccinated cohort, antibody assays that target the nucleocapsid protein or envelope protein are appropriate for diagnosing COVID-19 infection.
In summary, within 14 days of symptom onset, the positivity rate of SARS-CoV-2 antibody testing was relatively low compared with 15 days after onset. During the late stage of infection, the sensitivities of pan-Ig, IgG and combination assays are very high. However, in the early stage, the sensitivities vary among the assays. The pan-Ig assays, the SuperFlex™ combination assay and the IgG and iFlash combination assays were more sensitive than the Elecsys pan-Ig assay approved under EUA by the FDA. For the diagnosis of COVID-19, antibody testing should be performed 15 days after onset. For epidemiological surveillance, assays with high sensitivity, even if in the early stage, should be used, such as SuperFlex_Ab, iFlash_IgG, and their combination. IgM assays were not suitable for these purposes.