Comparative overview of currently available SARS-CoV-2 antibody tests LIVE WEBINAR 16 JULY 2020 Dr. Jörg-M. Hollidt
Different targets & different results: Exemplary evaluation of screening data Overview of available SARS-CoV-2 antibody tests Precise consideration - theoretical & experience- based reflection on specificity and sensitivity New targets & combinations - the way leading to a reliable result! in.vent Diagnostica GmbH | Dr. J.-M. Hollidt | SARS-CoV-2 antibody tests 16.07.2020 | 2
The Mission Targeted Procurement of Human Bio-Materials A precise assay needs precise samples. in.vent Diagnostica GmbH | Dr. J.-M. Hollidt | SARS-CoV-2 antibody tests 16.07.2020 | 3
in.vent – Competences certified according to: DIN EN ISO 9001 & DIN EN ISO 13485 • own Donation Center in Hennigsdorf for donors and patients • own manufacturing site for controls and calibrators • ICS : in.vent clinical services for IVD • own IVD R & D Unit ELISA, rapid tests, coated tubes: proof of principle / transfer to production • extensive experience in procuring, handling, processing, storage and logistics of human Bio-Materials • in.vent produces controls and calibrators for IVD manufacturer • 50+ highly qualified employees in.vent Diagnostica GmbH | Dr. J.-M. Hollidt | SARS-CoV-2 antibody tests 16.07.2020 | 4
Meeting the demands in.vent procures: inquiry to in.vent Any kind of human Bio-Material • Large volumes and pools • samples are in our biorepository Normal and disease state • yes no Clinically defined samples • Cohorts and panels • picking of start of a samples and procurement shipment to in.vent produces: project customer Controls / Calibrators • shipment of Proficiency tests • samples to customer in.vent Diagnostica GmbH | Dr. J.-M. Hollidt | SARS-CoV-2 antibody tests 16.07.2020 | 5
SARS-CoV-2 Pathogen of the global COVID-19 outbreak 2019/2020 • Enveloped positive single strand RNA virus: (+)ssRNA-virus • Belongs to the family Coronaviridae (crown-like appearance under electron microscope) • Spike protein (S) Lipid bilayer Spike: Viral receptor protein for adhesion to the host cell Membrane protein (M) Nucleocapsid protein (N) Capsid: protein coat around genome Envelope protein (E) ssRNA Structure of Coronavirus (09.07.2020) in.vent Diagnostica GmbH | Dr. J.-M. Hollidt | SARS-CoV-2 antibody tests 16.07.2020 | 6
Infection cycle of SARS-CoV-2 2/3 of 5´genome encodes polyproteins • which are cleaved into 16 non-structural proteins → forming replication complex 3´end of genome encodes 4 structural • proteins: spike (S), envelope (E), matrix/membrane (M), nucleocapsid (N) Attachment and fusion of SARS-CoV-2 with host cell by spike protein (source, 09.07.2020). angiotensin converting enzyme 2 receptor in.vent Diagnostica GmbH | Dr. J.-M. Hollidt | SARS-CoV-2 antibody tests 16.07.2020 | 7
Variety of coronaviruses Highly pathogenic zoonotic viruses ➢ SARS-CoV: 2002, 8.098 infected people, 774 death ➢ MERS-CoV: 2012, 2.494 infected people, 858 death Low-pathogenic common coronaviruses ➢ HCoV-OC43 ➢ HCoV-HKU1 infection rates of 40%-80% ➢ HCoV-NL63 Rinolophus sinicus (10.07.2020) ➢ HCoV-229E in.vent Diagnostica GmbH | Dr. J.-M. Hollidt | SARS-CoV-2 antibody tests 16.07.2020 | 8
Sequence identity between corona viruses Nucleocapsid protein 90.5 % sequence identity between SARS-CoV-2 and SARS-CoV • (97.2 % sequence similarity) Primary amino acid sequence of the N protein from SARS-CoV (ORF9a, NP_828858.1) and SARS-CoV-2 (ORF9, BCA87368.1) (source, 14.07.2020). in.vent Diagnostica GmbH | Dr. J.-M. Hollidt | SARS-CoV-2 antibody tests 16.07.2020 | 9
Sequence identity between corona viruses The spike protein of SARS-CoV-2 shares a high amino acid sequence identity with the other corona viruses. SARS-CoV-2 and SARS-CoV Spike protein: • 76 % sequence identity S2 subunit: 88 % sequence idnetity • → Sequence and conformational conservation suggests cross-reactivity of neutralizing Antibodies Ribbon diagrams of SARS-CoV-2 S (A) and SARS-CoV S (D) ectodomain and demonstration of S1 (B, E) and S2 subunits (C, F) (source, 10.07.2020). in.vent Diagnostica GmbH | Dr. J.-M. Hollidt | SARS-CoV-2 antibody tests 16.07.2020 | 10
Dependence of post translational modifications Glycosylation of SARS-CoV-2 Spike protein influences: Folding • Viral pathogenesis • Priming by host proteases • Antibody recognition • SARS-CoV-2 S comprise 22 N-linked glycosylations per protomer → at least 17 occupied Glycosylation of SARS-CoV-2 pike S protein (source, 10.07.2020). in.vent Diagnostica GmbH | Dr. J.-M. Hollidt | SARS-CoV-2 antibody tests 16.07.2020 | 11
Antigens of SARS-CoV-2 Several antigenes of SARS-CoV-2 are available: Nucleocapsid protein • Spike protein • Full-length S1 subunit • Search entries : „SARS -CoV- 2“; „ protein “ S1 N-terminal domain • 282 hits at BIOZOL Diagnostics Receprot binding domain • • Fulll-length S2 subunit • 241 hits at antibodies-online • Ectodomain (EDC) • Differing in label, application, purity, Envelope protein • modifications, origin of expression, reactivity […] Membrane protein • Non structural protein (NSP) 1, 2, 5, 7, 8, • 9, 10, 13, 14, 15 […] • in.vent Diagnostica GmbH | Dr. J.-M. Hollidt | SARS-CoV-2 antibody tests 16.07.2020 | 12
Seroconversion/ Immunstatus SARS-CoV-2 is different: SARS-CoV-1: • IgG detectable for 4-5 month ➢ Decreasing 2-3 years after ➢ seroconversion MERS-CoV: • Neutralizing Abs persisted up ➢ to 34 month after recovery Estimated Variation over time in diagnostic tests for detection of SARS-CoV-2 (source, 10.07.2020) in.vent Diagnostica GmbH | Dr. J.-M. Hollidt | SARS-CoV-2 antibody tests 16.07.2020 | 13
Diagnostics Company Ab class Antigen certification Company Ab class Antigen certification Abbott Diagnostics IgG N CE-IVD Assure Tech IgG, IgM N, S1 CE-IVD A. Menarini Diagnostics IgG, IgM No information CE-IVD Augurix IgG, IgM N, S1 CE-IVD Bestbion dx GmbH IgG, IgM, No information CE-IVD CeGaT IgG S1 CE-IVD IgA IBL International IgG, IgM, IgA Full-length N CE-IVD Bio-Rad Laboratories GmbH IgG, IgM, N CE-IVD Bio-Rad Total Ig N CE-IVD IgA FDA-EUA* DiaSorin Deutschland IgG S1, S2 CE-IVD Autobio IgG, IgM S FDA-EUA* GmbH FDA-EUA* Babson Diagnostics IgG S FDA-EUA* EUROIMMUN Medizinische IgG, IgA; S1; CE-IVD Labordiagnostika AG IgG, IgM N (diagnostic FDA-EUA* Biohit Healthcare IgG, IgM N FDA-EUA* relevant epitops) Cellex, Inc. IgG, IgM N, S FDA-EUA* GA Generic Assays GmbH IgG, IgM; Combind N+S CE-IVD IgG+ N, S1, S2 Emory Medical Laboratories IgG RBD FDA-EUA* Medac GmbH Diagnostika IgG, IgM N, S CE-IVD Hangzhou Biotest Biotech IgG, IgM RBD FDA-EUA* MIKROGEN GmbH IgG, IgA N CE-IVD Hangzhou Laihe Biotech IgG, IgM S FDA-EUA* Ortho Clinical Diagnostics Total Ig, S1 CE-IVD Healgen IgG, IgM S1 FDA-EUA* IgG FDA-EUA* InBios IgG S FDA-EUA* Roche Diagnostics IgG, IgM N CE-IVD, Mount Sinai Hospital IgG RBD FDA-EUA* Deutschland GmbH FDA-EUA* Siemens Healthcare Total Ig RBD FDA-EUA* Institut Virion/Serion IgG, Ectodomain CE-IVD Diagnostics GmbH IgM, IgA N, Ectodomain Vibrant America Clinical Labs IgG, IgM S1, RBD, S2, N FDA-EUA* VIROTECH Diagnostics IgG, IgM, N CE-IVD GmbH IgA Wadsworth Center, NY State Total Ig Full-length N FDA-EUA* Department of Health (from SARS-CoV-1) Aidian Germany GmbH IgG, IgM CE-IVD […] *FDA-EUA: Emergency Use Authorization by FDA Sources unless otherwise deposited: U.S. Food & Drug Administration; Trillium Diagnostik (14.07.2020) in.vent Diagnostica GmbH | Dr. J.-M. Hollidt | SARS-CoV-2 antibody tests 16.07.2020 | 14
Diagnostics 331 regulatory authorized diagnostic tests → 164 serological assays (total 230) Total serological assays 31 authorized serological assays 6 8 3x S1, 66 11 12 1x S1, S2 4x whole S 4x RBD 138 26 14 11 CE mark (approval to sell in Europe) Spike protein IgG IgG, IgM total Ig FDA-Emergency Use Authorization Nucleocapsid protein FDA do not distribute N, S combined Serological diagnostics overview (source; 14.07.2020) in.vent Diagnostica GmbH | Dr. J.-M. Hollidt | SARS-CoV-2 antibody tests 16.07.2020 | 15
Characteristics of selected tests Company AG Principle Cut-off Sensitivity Specificity Interferences Diasorin S1, Magnetic < 12 AU/mL 97.9 % 98.5 % (1000 No cross reactivity: beads source S2 negative; (48 samples taken blood donors) HuCoV OC43, NL63, coated with ≥ 15 AU/ mL > 15 days) 229E, MERS-CoV (1-2 S1/S2 positive samples) Roche NP Double- COI ≥ 1 99.5 % 99.8 % No cross reactivity: antigen- source = reactive (185 samples of (6.305 lab. HuCoV OC43, HKU1, sandwich, 103 PCR positive Routine; 4.148 229E CLIA patients ; ≥14days) blood donors < 15 % signal changes: IBL NP ELISA 10 NovaTec 9-11 days 40 % 99.24 % (133 Albumin, Bilirubin, Units (NTU) source (N=10) blood donors) Cholesterol, Hg, ≥12days 100 % Triglycerides (3 samples); (N=9) no cross-reactivity: 14 paramters No crossreactions: GA S1, ELISA 1 Binding >10 days 97.2 % 100 % (1000 HSV, EBV, CMV, HCV, HIV, Generic S2, Index (BI) IgG; 94.4 % IgM blood donors, HBsAg, Syphilis, Hg, Assays NP (N=36) lab. routine) Bilirubin, Triglyceride, Serum proteins, RF , etc. in.vent Diagnostica GmbH | Dr. J.-M. Hollidt | SARS-CoV-2 antibody tests 16.07.2020 | 16
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