Erytra blood group analyser and kode technology testing of SARS‐CoV‐2 antibodies among convalescent patients and vaccinated individuals

Abstract Surveillance of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) pandemic requires tests to monitor antibody formation and prevalence. We detected SARS‐CoV‐2 antibodies using red cells coated by Kode technology with short peptides derived from the SARS‐CoV‐2 spike protein (SP). Such modified red cells, called C19‐kodecytes, can be used as reagent cells in any manual or automated column agglutination assay. We investigated the presence of SARS‐CoV‐2 antibodies in 130 samples from COVID‐19 convalescent plasma donors using standard manual technique, two FDA‐authorized enzyme‐linked immunosorbent assay (ELISA) assays and a virus neutralisation assay. The sensitivity of the C19‐kodecyte assay was 88%, comparable to the anti‐SP and anti‐nucleocapsid protein (NCP) ELISAs (86% and 83%) and the virus neutralisation assay (88%). The specificity of the C19‐kodecyte assay was 90% (anti‐SP 100% and anti‐NCP 97%). Likewise, 231 samples from 73 vaccinated individuals were tested with an automated analyser, and we monitored the appearance and persistence of SARS‐CoV‐2 antibodies. The C19‐kodecyte assay is a robust tool for SARS‐CoV‐2 antibody detection. Automated blood group analyser use enables large‐scale SARS‐CoV‐2 antibody testing for vaccination monitoring in population surveys.


INTRODUCTION
In the COVID-19 pandemic, tests for virus ribonucleic acid (RNA) or virus particles enable the detection and isolation of infected individuals. The proportion of the population carrying antibodies following either infection or vaccination determines the herd-immunity.
How long protective antibodies persist after infection or vaccination remains to be determined. Large-scale population screens will provide this valuable information and facilitate the surveillance during the pandemic.
Many platforms for SARS-CoV-2 antibody testing have been launched [1], typically requiring specialized liquid handling and reader devices for result evaluation. We recently developed C19-kodecyte reagent red cells suitable for routine manual and automated assays with the antiglobulin techniques available in most blood bank and hospital laboratories [2,3]. C19-kodecyte reagent red cells can be prepared The results were compared to established enzyme-linked immunosorbent assay (ELISA) and a plaque reduction neutralisation assay [1]. In addition, we transferred the C19-kodecyte assay onto an automated blood group analyser and evaluated 231 samples from a vaccination monitoring study.

COVID-19-convalescent donor and control samples
Serum samples were sourced from blood donors who had recovered from mild to moderate PCR-confirmed COVID-19 disease and assessed as donors for convalescent plasma for a randomized prospective trial for treatment of patients with severe COVID-19 (CAPSID; EudraCT no. 2020-001310-38; ClinicalTrials.gov Identifier NCT04433910). All 130 samples were tested with the Euroimmun ELISA for antibodies directed against the SP and for antibodies against the nucleocapsid protein (NCP). In addition, 88 of these samples had been tested with the SARS-CoV-2 plaque reduction neutralisation test (PRNT) [1,4] which detects the reduction of wild-type virus-induced cell culture plaques. The results of the PRNT are given as the titer of sample at which a reduction of the plaques by 50% (PRNT50) or 90% (PRNT90) is observed. For the present study we used the PRNT50 results.
For negative controls, 38 serum samples were obtained from healthcare workers and their dependents (not known to have had  or been vaccinated). Eleven of these control samples were included in a recently published study [1].

2.2
Plasma samples from SARS-CoV-2 vaccination screening programme Informed consent was obtained, and individuals were tested for antibodies against SARS-CoV-2 prior to and after vaccination. This study was approved by the ethics board of the University of Ulm (no. 488/20).

C19-kodecyte assay
Serum samples from COVID-19 convalescent donors and controls were manually tested using Grifols DG antiglobulin and saline cards   Figure 1. In all cases, the visual grading was consistent with the grading of the Erytra.

Statistics
The sensitivity of the assays was calculated as the proportion of conva- Receiver operating characteristic (ROC) curves were plotted using a ROC plot package [5] for the statistical software R. to the results of the C19-kodecyte assay. The sensitivity of the C19kodecyte assay was 88% (Table 1), compared to 86% for the anti-SP IgG ELISA, 83% for the anti-NCP IgG ELISA and 88% for the virus neutralization assay. The specificity of the C19-kodecyte assay was 90%, while specificity of the ELISA for detecting anti-SP IgG and anti-NCP IgG was 100% and 97%, respectively. For comparison of the assays, ROC curves were drawn (Fig. 4).

Comparison of the C19-kodecyte assay with the ELISA and the PRNT
C19-kodecyte assay reaction grades (Figure 1), which semi-correlate with antibody levels [6], were compared with ELISA optical densities.

Automation of the C19-kodecyte assay
A total of 231 blood samples from 73 vaccinated individuals were tested with the Erytra Automated System. The routine antibody screening programme was employed, which encompasses three reagent cells and an autocontrol. The automated grading was in accordance with the grading as defined in Figure 1, and none of the results were manually edited. Data on the participants are shown in Table 3.
The results of 26 study participants who had an initial negative first sample result and then became positive are shown in Figure 5.

DISCUSSION
In this study, we further investigated the novel C19-kodecyte assay beta-corona viruses [8][9][10]. However, this rate was similar to the 91% specificity rate reported previously for the Grifols platform [2,7]. In a recent study, the specificity of the C19-kodecyte assay was found to be 96.3% [3]. The samples from negative controls had not been tested with the virus neutralization assay, so a comparison was not possible.
Although the majority of convalescent samples reacted positive with all three assays, there were also 31 samples that unexpectedly reacted negative with one or more assays. Eight samples were negative with all three assays suggesting that the level of antibodies in these samples were below the detection threshold. C19-kodecytes reacted positive with 15 of these 23 discordant samples (compared with 13/23 for anti-SP ELISA and 9/23 for anti-NCP ELISA) and therefore was unable to detect antibody in only eight samples that were positive by either or both of the ELISA assays.
This divergent reactivity of the C19-kodecyte assay with the ELISA assays has two major probable causes. First, the polyclonal antibody response after infection can differ between individuals, that is, one individual does not produce antibodies against the same epitopes of the virus as another individual. Therefore, individuals lacking or having lower levels of the antibodies specific for the target used in a specific assay may react negative, while reacting positive with another assay bearing a different target. Secondly, it should be noted that it is the cumulative result of all bound antibodies which is measured in an assay.
The C19-kodecyte assay only utilises two linear mono epitopes (MEps) from a domain of the SP which is located closely to the virus membrane [11], whereas the ELISA assays utilise the S1 domain of the SP, or a modified NCP, respectively. ELISA assays with large recombinant proteins are therefore representative of many linear and conformational For surveillance and control of the SARS-CoV-2 pandemic, it is necessary to know the current immunity status of the population. Early data on SARS-CoV-2 immunity suggested a rapid decline of the antibody levels [13,14] raising questions on the risk of re-infection of convalescents, while others found longer lasting antibody persistence [15].
Testing of large numbers of convalescents and vaccinated people for the collection of large data is required in order to draw meaningful con- As the reagent costs for the preparation of the kodecytes are below 0.10 € per test, and implementation is easy, this assay is potentially a valuable tool for the efforts of monitoring population immunity status in the SARS-CoV-2 pandemic.  Stephen M. Henry https://orcid.org/0000-0002-9946-8441