key: cord-261718-zqoggwnk authors: Pietschmann, Jan; Vöpel, Nadja; Spiegel, Holger; Krause, Hans-Joachim; Schröper, Florian title: Brief Communication: Magnetic Immuno-Detection of SARS-CoV-2 specific Antibodies date: 2020-06-03 journal: bioRxiv DOI: 10.1101/2020.06.02.131102 sha: doc_id: 261718 cord_uid: zqoggwnk SARS-CoV-2 causes ongoing infections worldwide, and identifying people with immunity is becoming increasingly important. Available point-of-care diagnostic systems as lateral flow assays have high potential for fast and easy on-site antibody testing but are lacking specificity, sensitivity or possibility for quantitative measurements. Here, a new point-of-care approach for SARS-CoV-2 specific antibody detection in human serum based on magnetic immuno-detection is described and compared to standard ELISA. For magnetic immuno-detection, immunofiltration columns were coated with a SARS-CoV-2 spike protein peptide. SARS-CoV-2 peptide reactive antibodies, spiked at different concentrations into PBS and human serum, were rinsed through immunofiltration columns. Specific antibodies were retained within the IFC and labelled with an isotype specific biotinylated antibody. Streptavidin-functionalized magnetic nanoparticles were applied to label the secondary antibodies. Enriched magnetic nanoparticles were then detected by means of frequency magnetic mixing detection technology, using a portable magnetic read-out device. Measuring signals corresponded to the amount of SARS-CoV-2 specific antibodies in the sample. Our preliminary magnetic immuno-detection setup resulted in a higher sensitivity and broader detection range and was four times faster than ELISA. Further optimizations could reduce assay times to that of a typical lateral flow assay, enabling a fast and easy approach, well suited for point-of-care measurements without expensive lab equipment. The new Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), is causing ongoing 31 worldwide infections, leading to an unprecedented pandemic. According to World Health 32 Organization (WHO), it is estimated that up to 82% of people with coronavirus disease 19 (COVID-33 19) are not aware that they are/were infected due to no or very mild symptoms [1] . 34 symptoms can be noted comparable to common cold cough, rhinitis or fever up to harsh symptoms, At a later point, serological assays would also be required to prove and monitor effectivity of 43 vaccination and longevity of the obtained immunity. Fast, cheap and easily applicable on-site testing 44 solutions will thus become increasingly important, but currently only few rapid test systems are 45 available. Lateral-flow-detection (LFD) approaches are easy to handle and results are gained after 10-46 15 min. However they are not quantitative, and their reliability, specificity and sensitivity is much 47 worse than that of lab-based assay formats based on enzyme-linked immunosorbent assay (ELISA). In particular, specificity is a major challenge at currently available serological antibody tests. This 49 3 depends to a large extent on the antigen used in the test assays. Enveloped positive-stranded RNA 50 SARS-CoV-2 coronaviruses consist of five structural proteins, the spike glycoprotein (S), envelope 51 protein (E), membrane protein (M), the nucleocapsid protein (N) and a hemagglutinin esterase (HE). The S-protein, a complex folded glycoprotein comprising two regions, S1 and S2, exhibits the highest 53 immunogenicity, has the most important role in host interaction, especially cell entry, and is also the 54 main target for neutralizing antibodies [5] . The proteins M, E and HE are only weakly immunogenic 55 and less suitable as targets for antibody diagnosis. Although the N protein is immunodominant, it is 56 not suitable for the specific analysis of the immune response against SARS-CoV-2 viruses due to its 57 high cross-reactivity with antibodies targeting related coronavirius strains [6, 7] . The company 58 Euroimmun AG, Lübeck, Germany offers two ELISA kits using a genetically modified N-protein 59 variant, which enables a more specific detection of antibodies already ten days after infection. However, for highly specific detection of immune response against SARS-CoV-2, typically the S1 61 subunit of S-protein should be used. Currently, only few vendors offer these specific ELISA formats 62 using the S1 subunit of S-protein for specifically detecting SARS-CoV-2 antibodies [8, 9] . Nevertheless, specific, sensitive and quantitative rapid tests applicable for a decentralized point-of-64 care (PoC) analysis are currently not available. Magnetic Immuno-Detection (MInD) could be a powerful tool for PoC assay performance. MInD As reference method to our PoC MInD approach, a typical laboratory-based ELISA was performed 173 (Fig 1) . After coating of ELISA microtiter plate with SARS-CoV-2 spike protein peptide and blocking 174 8 with BSA, SARS-CoV-2 spike protein peptide specific antibody was diluted in the range from 175 1.22 ng·ml -1 to 5000 ng·ml -1 in PBS-buffer or serum and applied into wells. After addition of 176 biotinylated GaR and subsequent labelling with streptavidin-AP, the ELISA plate was read out at 177 405 nm and obtained measuring values were used to generate calibration curves for SARS-CoV-2 178 specific antibody concentrations in PBS (Fig 1, black curve) and in human serum samples (Fig 1, red 179 curve). Blank values determined in PBS and serum were 0.085 AU ± 0.005 AU and Same calibration measurements employing dilutions of SARS-CoV-2 specific antibody were 211 done with our PoC MInD-based setup (Fig 2 and 3) . Comparable to laboratory-based ELISA, the same 212 dilutions of SARS-CoV-2 spike protein peptide specific antibody in PBS-buffer (Fig 3, black In this proof-of-concept experiment, a commercially available SARS-CoV-2 spike protein 267 peptide with corresponding antibody was used. If using this peptide for testing of patient samples, Funding: The author received no specific funding for this work. Acknowledgments: The authors would like to thank Max Schubert for his helpful advices and support given in 308 discussions. 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