Dynamic Assay for Profiling Anti-SARS-CoV-2 Antibodies and Their ACE2/Spike RBD Neutralization Capacity

Abstract

Serological assays have been widely employed during the coronavirus disease 2019 (COVID-19) pandemic to measure antibody responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to track seroconversion in populations. However, currently available assays do not allow determination of neutralization capacity within the assay protocol. Furthermore, commercial serology assays have a high buy-in cost that is inaccessible for many research groups. We have replicated the serological enzyme-linked immunosorbent assay for the detection of SARS-CoV-2 antibody isotypes, developed at the Icahn School of Medicine at Mount Sinai, New York. Additionally, we have modified the protocol to include a neutralization assay with only a minor modification to this protocol. We used this assay to screen local COVID-19 patient sera (n = 91) and pre-COVID-19 control sera (n = 103), and obtained approximate parity with approved commercial anti-nucleoprotein-based assays with these sera. Furthermore, data from our neutralization assay closely aligns with that generated using a spike-based pseudovirus infection model when a subset of patient sera was analyzed.

Both COVID-19 infection and vaccination induce high-affinity cross-clade responses to SARS-CoV-2 variants

“…Our observation was corroborated by repeating the measurements using the same IgG4-specific secondary antibody as well as by using the same clone from an alternative source (including differences in concentrations and storage buffers), which makes us confident that the results presented are valid. Moreover, we presented one of the most comprehensive cross-validations to date of all secondary antibodies -widely used in research on SARS-CoV-2 (Crowley et al, 2021;Fraley et al, 2021;Kim et al, 2021;Lee et al, 2021;Newell et al, 2021;Phelan et al, 2021;Pullen et al, 2021;Yates et al, 2021) and in other fields (Minassian et al, 2021;Sanchez Vargas et al, 2021;Tschismarov et al, 2021;Jennewein et al, 2022;Toney et al, 2022) -employed in this study and have confirmed their specificity to their target. We have noted that the sensitivity of the anti-human IgG1 antibody used can benefit from a higher concentration; however, it would come at a cost of compromising its specificity as the antibody shows cross-reactivity to other IgG subtypes at slightly increased concentrations, therefore, presenting a less favorable sensitivity-to-specificity profile than the other secondary antibodies.…”

Both COVID-19 infection and vaccination induce high-affinity cross-clade responses to SARS-CoV-2 variants

“…Our observation was corroborated by repeating the measurements using the same IgG4-specific secondary antibody as well as by using the same clone from an alternative source (including differences in concentrations and storage buffers), which makes us confident that the results presented are valid. Moreover, we presented one of the most comprehensive cross-validations to date of all secondary antibodies -widely used in research on SARS-CoV-2 (Crowley et al, 2021;Fraley et al, 2021;Kim et al, 2021;Lee et al, 2021;Newell et al, 2021;Phelan et al, 2021;Pullen et al, 2021;Yates et al, 2021) and in other fields (Minassian et al, 2021;Sanchez Vargas et al, 2021;Tschismarov et al, 2021;Jennewein et al, 2022;Toney et al, 2022) -employed in this study and have confirmed their specificity to their target. We have noted that the sensitivity of the anti-human IgG1 antibody used can benefit from a higher concentration; however, it would come at a cost of compromising its specificity as the antibody shows cross-reactivity to other IgG subtypes at slightly increased concentrations, therefore, presenting a less favorable sensitivity-to-specificity profile than the other secondary antibodies.…”

Severe Acute Respiratory Syndrome Coronavirus 2 Antibody Response After Heterologous Immunizations With ChAdOx1/BNT162b2 in End-Stage Renal Disease Patients on Hemodialysis

“…Nevertheless, natural infection and vaccinations produced a polyclonal antibody response targeting several regions of the spike protein ( 22 ). Moreover, a recent study has shown that mRNA vaccine-induced spike protein-specific antibodies continue to drive Fc effector functions, suggesting a capacity for extra-neutralizing antibodies to contribute to disease control ( 23 ). Thus, it is likely that the current serologic testing could reflect the humoral response against COVID-19 well.…”

Intranasal COVID-19 vaccine induces respiratory memory T cells and protects K18-hACE mice against SARS-CoV-2 infection

“…Antibodies in serum, lung and nasal wash of immunized mice were assessed for inhibition of binding of SARS-CoV-2 Spike to human ACE-2 as a surrogate of virus neutralization. The protocol was modified from that reported by Phelan et al 62 . High binding ELISA plates (Cruinn) were coated with 50 µL per well of Spike trimer (1 µg/ml) for 2 h at room temperature.…”

Both COVID-19 infection and vaccination induce high-affinity cross-clade responses to SARS-CoV-2 variants

“…Our observation was corroborated by repeating the measurements using the same IgG4-specific secondary antibody as well as by using the same clone from an alternative source (including differences in concentrations and storage buffers), which makes us confident that the results presented are valid. Moreover, we presented one of the most comprehensive cross-validations to date of all secondary antibodies -widely used in research on SARS-CoV-2 (Crowley et al, 2021;Fraley et al, 2021;Kim et al, 2021;Lee et al, 2021;Newell et al, 2021;Phelan et al, 2021;Pullen et al, 2021;Yates et al, 2021) and in other fields (Minassian et al, 2021;Sanchez Vargas et al, 2021;Tschismarov et al, 2021;Jennewein et al, 2022;Toney et al, 2022) -employed in this study and have confirmed their specificity to their target. We have noted that the sensitivity of the anti-human IgG1 antibody used can benefit from a higher concentration; however, it would come at a cost of compromising its specificity as the antibody shows cross-reactivity to other IgG subtypes at slightly increased concentrations, therefore, presenting a less favorable sensitivity-to-specificity profile than the other secondary antibodies.…”

Severe Acute Respiratory Syndrome Coronavirus 2 Antibody Response After Heterologous Immunizations With ChAdOx1/BNT162b2 in End-Stage Renal Disease Patients on Hemodialysis

“…Nevertheless, natural infection and vaccinations produced a polyclonal antibody response targeting several regions of the spike protein ( 22 ). Moreover, a recent study has shown that mRNA vaccine-induced spike protein-specific antibodies continue to drive Fc effector functions, suggesting a capacity for extra-neutralizing antibodies to contribute to disease control ( 23 ). Thus, it is likely that the current serologic testing could reflect the humoral response against COVID-19 well.…”

Intranasal COVID-19 vaccine induces respiratory memory T cells and protects K18-hACE mice against SARS-CoV-2 infection

“…Antibodies in serum, lung and nasal wash of immunized mice were assessed for inhibition of binding of SARS-CoV-2 Spike to human ACE-2 as a surrogate of virus neutralization. The protocol was modified from that reported by Phelan et al 62 . High binding ELISA plates (Cruinn) were coated with 50 µL per well of Spike trimer (1 µg/ml) for 2 h at room temperature.…”

Both COVID-19 infection and vaccination induce high-affinity cross-clade responses to SARS-CoV-2 variants

“…Our observation was corroborated by repeating the measurements using the same IgG4-specific secondary antibody as well as by using the same clone from an alternative source (including differences in concentrations and storage buffers), which makes us confident that the results presented are valid. Moreover, we presented one of the most comprehensive cross-validations to date of all secondary antibodies -widely used in research on SARS-CoV-2 (Crowley et al, 2021;Fraley et al, 2021;Kim et al, 2021;Lee et al, 2021;Newell et al, 2021;Phelan et al, 2021;Pullen et al, 2021;Yates et al, 2021) and in other fields (Minassian et al, 2021;Sanchez Vargas et al, 2021;Tschismarov et al, 2021;Jennewein et al, 2022;Toney et al, 2022) -employed in this study and have confirmed their specificity to their target. We have noted that the sensitivity of the anti-human IgG1 antibody used can benefit from a higher concentration; however, it would come at a cost of compromising its specificity as the antibody shows cross-reactivity to other IgG subtypes at slightly increased concentrations, therefore, presenting a less favorable sensitivity-to-specificity profile than the other secondary antibodies.…”

Severe Acute Respiratory Syndrome Coronavirus 2 Antibody Response After Heterologous Immunizations With ChAdOx1/BNT162b2 in End-Stage Renal Disease Patients on Hemodialysis

“…Nevertheless, natural infection and vaccinations produced a polyclonal antibody response targeting several regions of the spike protein ( 22 ). Moreover, a recent study has shown that mRNA vaccine-induced spike protein-specific antibodies continue to drive Fc effector functions, suggesting a capacity for extra-neutralizing antibodies to contribute to disease control ( 23 ). Thus, it is likely that the current serologic testing could reflect the humoral response against COVID-19 well.…”

Intranasal COVID-19 vaccine induces respiratory memory T cells and protects K18-hACE mice against SARS-CoV-2 infection

“…Antibodies in serum, lung and nasal wash of immunized mice were assessed for inhibition of binding of SARS-CoV-2 Spike to human ACE-2 as a surrogate of virus neutralization. The protocol was modified from that reported by Phelan et al 62 . High binding ELISA plates (Cruinn) were coated with 50 µL per well of Spike trimer (1 µg/ml) for 2 h at room temperature.…”