key: cord-0939116-075asnyp
authors: Moeller, Maria E.; Engsig, Frederik N.; Bade, Melanie; Fock, Jeppe; Pah, Pearlyn; Soerensen, Anna Louise; Bang, Didi; Donolato, Marco; Benfield, Thomas
title: Rapid Quantitative Point-Of-Care Diagnostic Test for Post COVID-19 Vaccination Antibody Monitoring
date: 2022-03-31
journal: Microbiol Spectr
DOI: 10.1128/spectrum.00396-22
sha: 2bb957e6061b4ba6b13e813cf69aac41ecb2cc65
doc_id: 939116
cord_uid: 075asnyp

Point-of-care (POC) quantification of antibody responses against SARS-CoV-2 spike protein can enable decentralized monitoring of immune responses after infection or vaccination. We evaluated a novel POC microfluidic cartridge-based device (ViroTrack Sero COVID-19 Total Ab) for quantitative detection of total antibodies against SARS-CoV-2 spike trimeric spike protein compared to standard laboratory chemiluminescence (CLIA)-based tests. Antibody responses of 101 individuals were measured on capillary blood, venous whole blood, plasma, and diluted plasma samples directly on the POC. Results were available within 7 min. As the reference, plasma samples were analyzed on DiaSorin LIAISON XL CLIA analyzer using LIAISON SARS-CoV-2 IgM, LIAISON SARS-CoV-2 S1/S2 IgG, and LIAISON SARS-CoV-2 TrimericS IgG assays. The Spearman rank’s correlation coefficient between ViroTrack Sero COVID-19 Total Ab and LIAISON SARS-CoV-2 S1/S2 IgG and LIAISON SARS-CoV-2 TrimericS IgG assays was found to be 0.83 and 0.89, respectively. ViroTrack Sero COVID-19 Total Ab showed high correlation between the different matrixes. Agreement for determination of samples of >230 binding antibody units (BAU)/mL on POC and CLIA methods is estimated to be around 90%. ViroTrack Sero Covid Total Ab is a rapid and simple-to-use POC test with high sensitivity and correlation of numerical results expressed in BAU/mL compared to those of a commercial CLIA assay. IMPORTANCE Serological testing is an important diagnostic support tool in the fight against COVID-19. So far, serological testing has been performed on either lateral flow assays, which perform only qualitatively and can be difficult for the individual to read, or standard laboratory assays, which are time- and resource-consuming. The purpose of the study was to evaluate the performance of a new POC microfluidic cartridge-based device based on immunomagnetic agglutination assay that can provide an accurate numerical quantification of the total antibodies within only 7 min from a single drop of capillary blood. We demonstrated a high level of correlation between the POC and the two CLIA laboratory-based immunoassays from Diasorin, thus allowing a potentially wider use of quantitative serology tests in the COVID-19 pandemic.

SARS-CoV-2-specific neutralizing antibodies upon infection and the focus of therapeutic and vaccine designs (4) (5) (6) (7) (8) . The correlates of protection are based on the specific level of SARS-CoV-2-specific neutralizing antibodies, acquired through vaccination or natural infection, that substantially reduces the risk of (re)infection (9, 10) .

In clinical trials, antibody production and cellular T cell responses have been measured for these candidate vaccines (11) (12) (13) (14) (15) . It has been shown that a large proportion of the individuals who mount immunoglobulin G (IgG) antibody responses against the viral S protein generate detectable neutralizing antibody responses (9) and that S protein binding assays correlate significantly with neutralization of wild-type SARS-CoV-2 virus (16) (17) (18) (19) (20) (21) (22) . Among the different subunits, the S protein in its trimeric form, when used in serology assays, has a high sensitivity (23) and specificity (22) .

Quantification of antibody responses and conversion rates of vaccinated populations can provide useful information not only to estimate the variety of vaccine responses and duration of protection but also to enhance vaccine immunogenicity, dosage optimization, amount, and time intervals (6, 24) . Therefore, it is inevitable that SARS-CoV-2 S-based assays play an essential role in vaccine efficacy monitoring.

Several quantitative IgG or total antibody tests based on enzyme-linked immunoassay (ELISA) or chemiluminescence-based instruments (CLIA) have been commercialized, and their performances have been evaluated in depth (25) (26) (27) . However, none of these methods are applicable for antibody quantification in decentralized settings. Standardization of the First WHO International Standard for anti-SARS-CoV-2 immunoglobulin (human; NIBSC code 20/136) has been introduced to allow for comparability between assay results. The International Standard is based on pooled human plasma from convalescent patients, which is lyophilized in ampules, with an assigned unit of 250 international units (IU) per ampule for neutralizing activity. For binding assays, a unit of 1,000 binding antibody units (BAU) per milliliter can be used to assist in the comparison of assays detecting the same class of immunoglobulins with the same specificity (28) .

The threshold of protection for anti-SARS-CoV-2 S protein antibodies acquired by vaccination is an object of research in the recent phase of the pandemic. Initial studies show that antibody levels associated with immunity against symptomatic COVID-19 infection measure about 150 to 200 BAU/mL, using the WHO International Standard (10, 29, 30) . High antibody titers have been reported as above 250 BAU/mL (31) . Recent studies show correlations among antibody titers 1 month postvaccination with the occurrence of breakthrough infections (32) .

A third vaccine shot has been shown to boost immune systems and block new emerging coronavirus variants (33) .

The aim of this study is to evaluate the performance of a new rapid quantitative pointof-care commercially available device from BluSense Diagnostics, based on the SARS-CoV-2 trimeric spike protein, ViroTrack Sero COVID-19 Total Ab, with two CLIA laboratory-based immunoassays from Diasorin, LIAISON SARS-CoV-2 S1/S2 IgG and LIAISON SARS-CoV-2 Trimeric S IgG assay. LIAISON SARS-CoV-2 Trimeric S IgG was chosen as comparison as it utilizes the same antigen of the ViroTrack test. The performance of the quantitative POC technology was evaluated on capillary and venous blood drawn from 101 hospitalemployed volunteers. A panel of precharacterized negative plasma and serum was further evaluated to determine the assay specificity.

Participant characteristics. A total of 101 participants were included. All characteristics can be found in Table 1 . A total of 47.5% of participants were between 20 and 39 years old, 44.65% were between 40 and 59 years old, and 8% were over 60 years of age. Of 101 participants, 93 had received two doses of vaccine, 1 had received one dose, and 7 were unvaccinated. Out of the double-vaccinated participants, 52 received their second dose between 2 and up to 5 weeks after the first dose, and 41 participants received their second dose at least 5 weeks (and up to 12 weeks) after the first dose. All the participants who received ChAdOx1 nCoV-19 (Astra Zeneca) as the first dose received BNT162b2 mRNA (Pfizer-BioNTech) as their second dose after at least 10 weeks. At the time of the study, all the participants who received BNT162b2 mRNA had received their second dose between 13 weeks to 29 weeks before the study, and all the participants who received ChAdOx1 nCoV-19 had received their second dose between 3 to 7 weeks before the study.

One capillary blood sample was not collected for one participant.

Comparison of assay performances in plasma. Plasma was analyzed by the POC device and the central lab CLIA-based assays. All vaccinated individuals had positive antibody titers with the ViroTrack system and the two Diasorin IgG assays. Two participants who were not vaccinated but had been previously infected by COVID-19 were negative by Diasorin TrimericS IgG but low positive (20 to 50 BAU/mL) with ViroTrack for all specimen types. Figure 1 shows the correlation between LIAISON SARS-CoV-2 S1/S2 IgG, LIAISON SARS-CoV-2 TrimericS IgG assays, LIAISON SARS-CoV-2 IgM, and ViroTrack Sero COVID-19 Total Ab for plasma samples.

A strong correlation between ViroTrack and the Diasorin TrimericS IgG and Diasorin S1/S2 IgG was observed. The Spearman rank's correlation coefficient was above 0.83 for all methods (Table 2) , and all the P values were below 10 226 . The highest correlation a All values are median and interquartile range. b Includes systemic and topical medicine (e.g., steroid nasal spray or inhalation). c One study subject identified with two occupations. d All vaccinated participants had received Pfizer-BioNTech (Comirnaty) as their second injection regardless of type of the first. e The day of test was noted as the last study day resulting in an uncertainty of 0 to 3 days. f One study subject was yet to have the second injection when participating.

with ViroTrack was by the Diasorin Trimeric S assay. The Diasorin M assay did not correlate with the other tested methods.

Comparison among capillary blood, venous blood, and plasma results. Having established the correlation between ViroTrack and the reference test methods, we investigated the correlation between different specimen types. Figure 2A and B show the results for different undiluted specimen types for values below 230 BAU/mL.

The correlation between the different specimen types measured by ViroTrack and the plasma samples using Diasorin TrimericS IgG for determination of above 230 BAU/mL is shown in Table 3 . The agreements are above 90% between all specimen types and both methods.

Quantitative results versus vaccination/previous infectious status. The quantitative results obtained by the POC device were analyzed according to vaccination and prior infection. Figure 3 shows a swarm plot of the data. For the nonvaccinated, previously infected, the median was 1,850.8 BAU/mL (interquartile range [IQR] 59.9; 3,641.7). For the non-previously infected who were vaccinated (two injections), we observe a lower antibody response (median: 2,686. 8 ). However, these participants had all received the second dose with BNT162b2 mRNA less than 50 days before the study, whereas the participants receiving two doses of BNT162b2 mRNA in most cases received the second dose more than 125 days before the time of the study. The time between the injections also varied for the two groups.

Several of the noninfected who received both doses of BNT162b2 mRNA showed values below 230 BAU/mL. For the previously infected, only participants without any vaccination had values below 230 BAU/mL. The capability of the assays to correctly quantify a high positive sample (e.g., .230 BAU/mL) can be important, as revealed by recent reports correlating between the antibody levels and the immunity against symptomatic COVID-19 infection (29, 30) .

The study included two nonvaccinated and noninfected participants (see Table 1 ). The two subjects tested negative by all methods and sample types. To determine the specificity of the assay, a panel of 699 negative samples were measured. It was composed of samples from healthy donors, samples collected prior to 2019, and samples positive for other viruses, bacteria, or potential cross-reactive substances. Data are reported in Table 4 and show a specificity of 99.7%.

To our knowledge, this is the first report comparing the numerical results of a rapid quantitative COVID-19 serology test with a reference CLIA method. There exist a few quantitative rapid serology tests with a reader based on fluorescence lateral flow tests already; however, so far, only qualitative rapid tests have been evaluated in literature (34) . Our study showed a statistically high level of correlation between the results from ViroTrack Sero COVID-19 Total Ab and those from the two CLIA laboratory-based immunoassays from Diasorin, LIAISON SARS-CoV-2 S1/S2 IgG and LIAISON SARS-CoV-2 Trimeric S IgG assay. The highest correlation, 0.94, was found for LIAISON SARS-CoV-2 Trimeric S IgG assay, which utilizes the same spike trimer antigen as ViroTrack Sero COVID-19 Total Ab.

Previous reports of other commercial spike protein-based IgG assays (ELISA or CLIA based) showed a lower level of correlation of around 0.7 to 0.8 (26) with or without standardization to BAU/mL units (25) . The high correlation was obtained even though the POC method measures total antibodies (IgG, IgM, and IgA) while the reference methods measured only IgG and/or receptor binding domain. The low influence of IgA and IgM antibodies may be explained by a low IgM concentration, a general correlation between IgA and IgG titers, or the predominance of IgG antibody class in vaccinated individuals. The agreement among specimen types was satisfactory. As described, the ViroTrack Sero COVID-19 Total Ab assay is embedded into a centrifugal microfluidics platform where blood is separated into plasma in the initial processing steps. This unique capability allows for the precise quantification without influence of factors such as hematocrit, enabling precise correlation with laboratory-based methods. To our knowledge, systematic studies comparing COVID-19 antibodies in different matrixes do not exist; however, preliminary studies show differences in rapid test results when capillary or venous blood is used (35) . We observed a higher antibody response for participants with ChAdOx1 nCoV-19 as the first dose and BNT162b2 mRNA as the second dose less than 50 days prior to the study compared to that of the participants who, in most cases more than 125 days prior to the study, received their second dose of their two doses from BNT162b2 mRNA. Furthermore, its known that heterologous boosting results in higher titers than homologous boosting (36, 37) .

A limitation of this study is represented by the fact that only two individuals were neither previously infected nor vaccinated; however, the large negative retrospective sample panel measured (699 samples) confirmed the high specificity of the product. In addition, in a previous study we showed that a first version of the POC test targeting the antibodies against SARS-COV-2 nucleocapsid protein had a higher specificity than ELISA-based methods (38) . Second, the study is limited, as an extra dilution step was necessary to extend the current dynamic range of the POC device which is currently not included in the product "instructions for use," and the dilution process performed in blood may have produced a different result. However, the data demonstrated that the device produced an accurate quantification of diluted plasma.

A general agreement between capillary blood, venous blood, and plasma from the same samples and techniques has been found, thus supporting the use of capillary blood on the POC device for precise decentralized antibodies monitoring postvaccination and responses after natural infection in countries where the use of vaccines is low or yet to come.

Among the vaccinated-only individuals, 17 (18%) had antibodies below 200 BAU/mL, but their median time from last vaccination to antibody did not differ from that of patients who had above 230 BAU/mL (136 days). This could be caused by several things, including unrecognized or undiagnosed infection with COVID-19, as none of our assays included antibodies against antigens/epitopes other than the spike protein and information on previous infection with SARS-CoV-2 relied solely on participant memory, which could introduce a bias. On the other hand, the main purpose of the study was to evaluate the performance of a new technology and our result should not be affected by this.

In conclusion, ViroTrack Sero COVID-19 Total Ab provides an accurate numerical quantification of the total antibodies against the spike protein trimer within 7 min from a single drop of capillary blood. Compared to rapid lateral flow tests detecting antibodies against different forms of the spike protein, the evaluated POC device provides a numerical result in a shorter time.

This capability can enable precise monitoring of antibodies amounts in facilities in various places, allowing a potentially wider use of quantitative serology tests in the COVID-19 pandemic.

Subjects and samples. All participants were staff, and included individuals were from most types of professions (e.g., doctors, nurses, physiotherapists, cleaning staff, etc.). Each study subject was asked to fill in a questionnaire using research electronic data capture ( Table 1 ). The study was approved by the Regional Ethics Committee of the Capital Region of Denmark (record no. H-20046624). The study was further approved by the Regional Data Protection Center (record no. P-2020-358).

Sample collection. Capillary and venous blood samples were collected from each of the study subjects. Venous blood, capillary blood, and plasma samples (diluted or undiluted) were analyzed with the ViroTrack Sero COVID-19 Total Ab. The plasma samples were further analyzed on a Diasorin LIAISON XL analyzer at the Department of Clinical Microbiology. Approximately 5 mL of venous blood was collected per subject in a blood collection tube (BD Vacutainer, UK) treated with potassium ethylenediaminetetraacetic acid (EDTA). Blood was processed following manufacturer's instruction and used for plasma separation. Briefly, blood was centrifuged for 15 min at 1,500 Â g and 20°C to obtain plasma.

For analyzing capillary blood, 20 mL was collected with a micropipette or a capillary pipette. Capillary blood was loaded immediately onto the ViroTrack Sero COVID-19 Total Ab cartridge after collection; product specifications allow for 3 min before starting the test. Venous blood was stored at room temperature after collection and to a maximum of 5 h before testing on the ViroTrack Sero COVID-19 Total Ab cartridge. Plasma was separated and stored at room temperature and to a maximum of 5 h before testing onto the ViroTrack Sero COVID-19 Total Ab cartridge. All plasma samples were stored at 280°C prior to Diasorin LIAISON XL analyzer testing, which took place 7 to 11 days after the study. To assess the specificity of the test, a panel of 699 negative plasma and serum samples were tested at COVID-19 or Anti-SARS-CoV-2 Antibody Point-Of-Care Test Microbiology Spectrum

BluSense Diagnostics as part of the product validation and CE marking certification process. The samples were previously characterized by the sample providers and/or collected before 2019. ViroTrack Sero COVID-19 Total Ab. The ViroTrack Sero COVID-19 Total Ab is a POC rapid test providing quantitative results within 7 min in the range of 10 to 230 BAU/mL from 20 mL blood, plasma, or serum. The test format is composed by a cartridge (ViroTrack Sero COVID-19 Total Ab) and a reader (BluBox).

The platform utilizes a centrifugal microfluidic platform together with an optomagnetic readout based on the agglutination of magnetic nanoparticles (IMA). In brief, 20 mL of sample is loaded on to the microfluidic cartridge, which is then inserted inside the reader (BluBox). In the case of whole blood, the red blood cells are separated from the plasma by centrifugal force. The separated plasma is subsequently resuspended in the prestored reagents on the cartridge (e.g., magnetic particles). The magnetic particles are functionalized with SARS-CoV-2 trimeric spike protein and agglutinates in a sample containing anti-spike antibodies. Incubating the particles in a homogeneous magnetic field speeds up the reaction kinetics of the agglutination (39, 40) . For optomagnetic detection, a uniaxial alternating magnetic field is applied which periodically aligns the agglutinated particle chains, which results in a modulation of the transmitted light proportional with the target concentration (41) . IMA does not require labeled secondary antibodies (38) .

The ViroTrack Sero COVID-19 Total Ab was calibrated to the First WHO International Standard for anti-SARS-CoV-2 immunoglobulin (code: 20/136), and the results were converted to binding antibody units per milliliter (BAU/mL) by the software up to 230 BAU/mL. Plasma samples with .230 BAU/mL were diluted 10 times in phosphate-buffered saline (PBS) and remeasured. Dilution (20 times and 40 times) was continued until a result below 230 BAU/mL was obtained. The final binding antibody units per milliliter was found by multiplying the dilution factor with the obtained result. During the study, four different BlueBox readers were used in parallel. Samples were loaded in different readers in a randomized order.

The same data are shown in the product instruction for use and used for CE certification.

Diasorin. The plasma samples were analyzed by chemiluminescence immunoassay (CLIA) for SARS-CoV-2 antibodies (IgM and IgG) targeting the subunits of the spike proteins S1 and S2 and the trimeric spike complex, including the receptor binding domain (S1-RBD). Samples were analyzed on the DiaSorin LIAISON XL analyzer using LIAISON SARS-CoV-2 IgM, LIAISON SARS-CoV-2 S1/S2 IgG, and LIAISON SARS-CoV-2 TrimericS IgG assays in accordance with the manufacturer's instructions on the specific assay. A negative anti-SARS-CoV-2 result was defined as IgM index of ,1.1 and IgG of ,12 antibody units (AU)/ mL, and a positive result was defined as an index value of $1.1 AU/mL for IgM and $15 AU/mL for IgG, respectively. Results for IgG of ,15 AU/mL and $2 AU/mL were reported as inconclusive.

A TrimericS IgG result was defined as negative with a value of ,13 AU/mL, and a positive result was defined as values of $13 AU/mL (equivalent to $33.8 BAU/mL). The LIAISON SARS-CoV-2 TrimericS IgG assay measures between 4.81 and 2,080 BAU/mL. A recent study demonstrated that the DiaSorin SARS-CoV-2 S1/ S2 IgG antibodies had a sensitivity of 96.2% and a specificity of 98.9% (42) , whereas the DiaSorin TrimericS IgG has been shown to have a higher sensitivity of 99.4% and a higher specificity of 99.8% (22) .

Statistical analysis. Spearman's rank correlation was measured to evaluate the agreement between the different assays and the different specimen types. Analysis and graphs were performed using PYTHON/R.

We thank Heike Ebermann from the Department of Microbiology, Copenhagen University Hospital -Amager and Hospital for excellent technical assistance and Laura Catalina Bohorquez for her help in drafting the paper introduction. 

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The authors declare a conflict of interest. P. Pah, M. Bade, J. Fock, and M. Donolato are employed at Blusense Diagnostics APS and have been part of the developing of ViroTrack Sero COVID-19 Total Ab. This work was supported by BluSense Diagnostics who provided the test kits for the study.