Microscopy‐based assay for semi‐quantitative detection of SARS‐CoV‐2 specific antibodies in human sera

Abstract

Emergence of the novel pathogenic coronavirus SARS‐CoV‐2 and its rapid pandemic spread presents challenges that demand immediate attention. Here, we describe the development of a semi‐quantitative high‐content microscopy‐based assay for detection of three major classes (IgG, IgA, and IgM) of SARS‐CoV‐2 specific antibodies in human samples. The possibility to detect antibodies against the entire viral proteome together with a robust semi‐automated image analysis workflow resulted in specific, sensitive and unbiased assay that complements the portfolio of SARS‐CoV‐2 serological assays. Sensitive, specific and quantitative serological assays are urgently needed for a better understanding of humoral immune response against the virus as a basis for developing public health strategies to control viral spread. The procedure described here has been used for clinical studies and provides a general framework for the application of quantitative high‐throughput microscopy to rapidly develop serological assays for emerging virus infections.

Prevalence of SARS-CoV-2 Infection in Children and Their Parents in Southwest Germany

“…Studies by our team and others found a slightly lower specificity when including borderline results (eg, 96.3% or 98.6%). 16,26 Given the low SARS-CoV-2 prevalence in the general population, when this study was performed, test combinations with specificities greater than 99% are required to obtain valid results. A combination of 2 different tests improves accuracy markedly and has recently been used in a large seroepidemiological study.…”

“…15 Here, we combined ELISA with immunofluorescence as complementary tests for all samples. 16 Moreover, we further analyzed discordant samples by additional tests to maximize both specificity and sensitivity. As shown in Table 2, both initial assays independently yielded 40% to 50% of false-positive results, but these could be eliminated by a combination of the 2 readouts.…”

“…A number of samples (n = 119) displayed borderline reactivity in ELISA testing or yielded discordant results between ELISA and immunofluorescence tests. 16 In these cases, at least 1 of the additional tests was used for final classification. As shown in Table 2, only a small proportion of the samples that had positive results in only 1 of the initial assays was finally confirmed as posi-tive (1 [3.8%] via ELISA only and 4 [8.5%] via immunofluorescence only); the confirmed proportion was also small for samples that had borderline results in ELISA tests and negative results in immunofluorescence tests (2 [4.8%]).…”

Advanced microscopy technologies enable rapid response toSARS‐CoV‐2 pandemic

“…Studies on SARS-CoV-2-induced cellular ultrastructural changes have been vital in understanding viral impact on infected host cells and have provided clues for potential therapeutic procedures (Cortese et al, 2020;Ogando et al, 2020;Wolff et al, 2020). Microscopy-based serological assays enabled specific, sensitive and quantitative assessment of SARS-CoV-2-specific antibodies and proved to be especially useful in situations where commercial assays are either not yet developed or in short supply due to a high demand (Pape et al, 2020). Moreover, the employment of microscopy-based virus neutralisation assays paved the way to fast vaccine development (Mulligan et al, 2020;Muruato et al, 2020).…”

“…The major disadvantage of using immunofluorescence microscopy in serology is its limited throughput capacity due to manual microscopy handling steps during image acquisition and sample evaluation based on visual inspection of micrographs which is subjective and not quantitative. These limitations have been addressed byPape et al, where a high-throughput microscopy-compatible procedure was developed together with a robust machine learning-based image analysis pipeline yielding an assay with slightly better specificity/sensitivity than a commercially available ELISA assay at the time(Pape et al, 2020). Microscopybased serological tests have been used in large population SARS-CoV-2 serology studies mainly as a confirmatory assay to provide increased specificity of detection in the setting of low antibody prevalence(Stringhini et al, 2020;Tönshoff et al, 2021).…”