LIPS method for the detection of SARS-CoV-2 antibodies to spike and nucleocapsid proteins

Abstract

Profiling antibodies to SARS-CoV-2 can help to assess potential immune response after COVID-19 disease. Luciferase IP system (LIPS) assay is a sensitive method for quantitative detection of antibodies to antigens in their native conformation. We here describe LIPS to detect antibody responses to SARS-CoV-2 spike (S) and nucleocapsid (N) proteins in COVID-19 patients. The antibodies targeted both S and N fragments and gave a high assay sensitivity by identifying 26 out of 26 COVID-19 patients with N antigen or with three protein fragments when combined into a single reaction. The assay correlated well with ELISA method and was specific to COVID-19 as we saw no reactivity among uninfected healthy controls. Our results show that LIPS is a rapid and measurable method to screen antibody responses against SARS-CoV-2 antigens.

Keywords: COVID-19; LIPS; SARS-CoV-2; antibodies; luciferase immunoprecipitation.

Figure 1

LIPS analysis of antibodies to SARS‐CoV‐2 antigens. S1, S2, and N genes were cloned in fusion with NanoLuc (Promega) gene and expressed in HEK293 cells. The cell lysates were incubated with plasma samples (in 1:40 dilution) and bound to Protein G Sepharose to capture antibody complexes with viral proteins. After the washing, luciferase substrate Nano‐Glo™ (Promega) was added and luminescence was measured in VICTOR X multilabel reader (PerkinElmer Life Sciences). Results are expressed as fold changes (FC) of luminescence units (LU) (FC = LU sample/average LU of five healthy control samples). The positive/negative discrimination level was set to the mean plus two standard deviations of the healthy control samples. The LIPS experiments were performed three times in three experimental replicates with 26 patients and 26 controls per experiment. The heatmap (D) shows average reactivities to three antigens in individual patients. The correlation of LIPS values between S1 and S2 (E), S1 and N (F), and S2 and N (G) antibody values. Triton X was added to the assays to test its impact on LIPS performance with S1 (H), S2 (I), and N (J) antigens. Mixing three antigens (S+N mix) in a single LIPS assay was correlated with the sum of the values from the three different assays (S1+S2+N) (K) and compared between patients and controls (L). The anti‐SARS‐CoV‐2 IgG ELISA (Euroimmun) was performed according to the manufacturer's instructions. Statistics were performed using unpaired Student's t‐test and Pearson correlation analysis in Graphpad Prism. ****p < 0.0001.