key: cord-0721939-nn7itiys
authors: Haljasmägi, Liis; Remm, Anu; Rumm, Anna Pauliina; Krassohhina, Ekaterina; Sein, Hanna; Tamm, Anu; Kisand, Kai; Peterson, Pärt
title: LIPS method for the detection of SARS‐CoV‐2 antibodies to spike and nucleocapsid proteins
date: 2020-07-06
journal: Eur J Immunol
DOI: 10.1002/eji.202048715
sha: 475a83f8650e7c729066406965d0354224337bde
doc_id: 721939
cord_uid: nn7itiys

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.

The plasma samples were incubated with lysates containing S1, S2 and N fusion protein solutions (0.5 -1 × 10 6 luminescence units; LU) for 1 h at RT. The Protein G Sepharose beads (25 µl of 4% suspension, Creative BioMart) were added and incubated at room temperature for 1 h in 96-well microfilter plates (Merck Millipore) to capture antibodies (in 1:40 dilution) and immune complexes to the beads. After the washing to remove unbound fusion proteins, luciferase substrate was added (Nano-Glo™ Luciferase Assay Substrate, Promega), and luminescence was measured in VICTOR X Multilabel Plate Reader (PerkinElmer Life Sciences). Results are expressed as fold changes (FC) of luminescence units (LU) (FC=LU sample/average LU of 5 healthy control samples). The positive/negative discrimination level was set to the mean plus 2 standard deviations of the healthy control samples. The experiments were performed three times in three experimental replicates. Statistics was performed using unpaired Student's t-test and Pearson correlation analysis in Graphpad Prism.

The Anti-SARS-CoV-2 IgG ELISA (Euroimmun Medizinische Labordiagnostika; Cat # EI 2668-9601 G) was performed according to the manufacturer's instructions. In semi-quantitative ELISA, IgG antibodies against SARS-CoV-2 S1 protein subunit S1 were detected. Briefly, 1:101 diluted plasma samples were added to wells coated with recombinant SARS-CoV-2 antigen and incubated for 60 minutes at 37 °C. Wells were washed three times followed by the addition of HRP-conjugated anti-human IgG and subsequent incubation for 30 minutes at 37 °C. Wells were washed three times again and a chromogen solution was added. Following 30 minutes of incubation at room temperature, the reaction was stopped and the resultant absorbance was read on a microplate reader at 450 nm with reference at 620 nm. A ratio between the extinction of the sample and calibrator on plate was calculated. According to the manufacturer's recommendations, a ratio <0.8 is considered negative, ≥0.8 and <1.1 borderline, and ≥1.1 positive.

Supporting Table 1 . Main characteristics, LIPS and ELISA results of each studied patient. Patient sex, age and plasma sampling day, SARS-CoV-2 antibody values in LIPS with three antigens and EUROIMMUNE ELISA assay optical density (OD) values and its ratios to controls are given. Supporting Figure 1 . Western blot for the detection of NanoLuc-S1, NanoLuc-S2 and NanoLuc-N fusion proteins. The cell lysates were run a single 10% polyacrylamide gel, transferred to PVDF membrane, which was cut into two after the transfer. Left side: transfected HEK293 cell lysates of NanoLuc-S1 (S1) and NanoLuc-S2 (S2) were probed with anti-Spike1 antibody (diluted 1:2000, GeneTex). Right side: transfected HEK293 cell lysates of NanoLuc-S1, NanoLuc-S2, NanoLuc-N (N) and NanoLuc were probed with anti-NanoLuc antibody (diluted 1:500, Promega). The membranes were further incubated with HRP-labeled antirabbit (for S1) and anti-mouse (for NanoLuc) (both from Jackson ImmunoResearch). The detected proteins are shown by asterisks. The molecular marker lanes are shown on both sides of the membrane. The anti-S1 antibody (on left side) detects NanoLuc-S1 protein as a double band at approximately 110-120 kDa. The anti-NanoLuc antibody detects NanoLuc-S1 protein at the same location albeit weaker than seen with anti-S1 antibody. NanoLuc-S2 and NanoLuc-N are seen as strong bands at 75-80kDa. NanoLuc is seen at 20kDa but also at 38-40kDa. The predicted molecular mass of the proteins without post-translational modifications are 100kDa (NanoLuc-S1), 62kDa (NanoLuc-S2) and 59kDa (NanoLuc-N).

Supporting Figure 2 . Empty NanoLuc vector without SARS-CoV-2 antigens was assayed with 4 Covid-19 and 3 control plasma samples. The NanoLuc (Promega) gene was 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 TGTTGTTAAC TTGTTTATTG CAGCTTATAA TGGTTACAAA TAAAGCAATA  1680  GCATCACAAA TTTCACAAAT AAAGCATTTT TTTCACTGCA TTCTAGTTGT GGTTTGTCCA  1740  AACTCATCAA TGTATCTTAA GGCGTAAATT GTAAGCGTTA ATATTTTGTT AAAATTCGCG  1800  TTAAATTTTT GTTAAATCAG CTCATTTTTT AACCAATAGG CCGAAATCGG CAAAATCCCT  1860  TATAAATCAA AAGAATAGAC CGAGATAGGG TTGAGTGTTG TTCCAGTTTG GAACAAGAGT  1920  CCACTATTAA AGAACGTGGA CTCCAACGTC AAAGGGCGAA AAACCGTCTA TCAGGGCGAT  1980  GGCCCACTAC GTGAACCATC ACCCTAATCA AGTTTTTTGG GGTCGAGGTG CCGTAAAGCA  2040  CTAAATCGGA ACCCTAAAGG GAGCCCCCGA TTTAGAGCTT GACGGGGAAA GCCGGCGAAC  2100  GTGGCGAGAA AGGAAGGGAA GAAAGCGAAA GGAGCGGGCG CTAGGGCGCT GGCAAGTGTA  2160  GCGGTCACGC TGCGCGTAAC CACCACACCC GCCGCGCTTA ATGCGCCGCT ACAGGGCGCG  2220  TCAGGTGGCA CTTTTCGGGG AAATGTGCGC GGAACCCCTA TTTGTTTATT TTTCTAAATA  2280  CATTCAAATA TGTATCCGCT CATGAGACAA TAACCCTGAT AAATGCTTCA ATAATATTGA  2340  AAAAGGAAGA GTCCTGAGGC GGAAAGAACC AGCTGTGGAA TGTGTGTCAG TTAGGGTGTG  2400  GAAAGTCCCC AGGCTCCCCA GCAGGCAGAA GTATGCAAAG CATGCATCTC AATTAGTCAG  2460  CAACCAGGTG TGGAAAGTCC CCAGGCTCCC CAGCAGGCAG AAGTATGCAA AGCATGCATC  2520  TCAATTAGTC AGCAACCATA GTCCCGCCCC TAACTCCGCC CATCCCGCCC CTAACTCCGC  2580  CCAGTTCCGC CCATTCTCCG CCCCATGGCT GACTAATTTT TTTTATTTAT GCAGAGGCCG  2640  AGGCCGCCTC GGCCTCTGAG CTATTCCAGA AGTAGTGAGG AGGCTTTTTT GGAGGCCTAG  2700  GCTTTTGCAA AGATCGATCA AGAGACAGGA TGAGGATCGT TTCGCATGAT TGAACAAGAT  2760  GGATTGCACG CAGGTTCTCC GGCCGCTTGG GTGGAGAGGC TATTCGGCTA TGACTGGGCA  2820  CAACAGACAA TCGGCTGCTC TGATGCCGCC GTGTTCCGGC TGTCAGCGCA 

Nucleocapsid gene fragment with BamHI and NotI restriction enzyme sites ID N gene; linear

GGATCCTAtc tgacaacggc cctcagaacc agcggaacgc tcctcggatc accttcggcg