Background
Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), first detected in Wuhan, China in December 2019 has become a global pandemic with approximately 108.2 million infections and 2.4 million deaths worldwide on 13 February 2021 [
1]. Infection is most commonly diagnosed by nucleic acid amplification tests (NAATs), often RT-qPCR, performed on nasopharyngeal and mid-turbinate swabs [
2]. Detection of antibodies to SARS-CoV-2 is however important for several purposes including: (i) confirming present or past infection, (ii) evaluating patients with negative NAATs who show characteristic COVID-19 symptoms, (iii) sero-epidemiological studies on COVID-19, (iv) assessing the development of antibody-mediated protective immunity, and (v) investigating immune response and immunopathology in COVID-19 [
3,
4]. The spike (S) and nucleocapsid (N) proteins of SARS-CoV-2 are commonly used target antigens in COVID-19 serological assays. The S protein is exposed on the outside of the virus membrane while N encapsulates viral RNA within the membrane envelope. S is composed of a N-terminal S1 region containing a receptor binding domain (RBD) which binds to the angiotensin-converting enzyme 2 receptor on host cells, and a C-terminal S2 region that subsequently mediates fusion between the viral and host cell membranes to allow the entry of viral RNA into the cell [
5].
Different platforms are available for serological testing in COVID-19. Lateral flow immunoassays are common point of care serological tests and produce results in <30mins. Enzyme Immunoassay (EIA) is the most frequently used serological method in clinical laboratories [
3,
4]. EIAs usually measure antibodies to single antigens in a clinical laboratory over a period of several hours, and have been important for assessing antibody responses in COVID-19 [
6,
7] and COVID-19 vaccine trials [
8].
We describe here for the first time the use of recombinant S1, S2, RBD and N proteins as antigens in multiplex line immunoblots (IBs) to detect antibodies in COVID-19 patients. The COVID-19 IgG and IgM IB assays detect IgG and IgM antibodies to S1, S2, RBD and N proteins in less than 3 h.
Discussion
The clinical sensitivity and specificity of the COVID-19 IB assays for IgM, IgG and IgG and/or IgM antibodies, estimated by pooling multiple samples from the same patient, meet the US recommendations for laboratory serological diagnostic tests [
11]. They fall short of the expected specificity of > 99.5% in the US for large sero-surveillance studies in the community where the prevalence is expected to be very low [
3]. However, it is possible that COVID-19 IBs may be useful for sero-epidemiological studies in specific populations with a high prevalence of COVID-19. Our results confirm other common observations in COVID-19 that IgM and IgG antibody levels vary with time after infection [
3,
4,
11]. This is expected because antibodies are produced with a lag period after infection, early IgM is later replaced with IgG antibodies and antibody levels in blood generally decrease with time after resolution of infection. Our findings suggest that determining both IgG and IgM antibodies early in an infection i.e. before about 10 days from onset of disease, and IgG antibodies later at about 8 weeks after infection, provide the best sensitivity for detecting antibody responses in COVID-19 IBs. However, more extensive testing of patient samples and other human sera as specificity controls will better establish the clinical diagnostic parameters of the COVID-19 IBs and the optimal times for their use after infection.
Results using pre-prepared IB membrane strips can be obtained in less than 3 h after serum or plasma collection, with minimal washing and reagent addition steps. Also, the assay yields visible signals that are stable for several weeks and readily interpreted relative to an internal calibrator. Furthermore, the IB assay can be adapted for detecting antibodies of other immunoglobulin classes, and in other relevant fluids, e.g. saliva and tears, which is important because the mucosal IgA and blood IgG and IgM antibody responses can differ significantly in COVID-19 [
12,
13]. The COVID-19 IB assay can also be easily expanded to include additional virus antigens. Fixed dilutions of patient sera (1:100 in the IgG and 1:50 in the IgM assays respectively) were used to obtain the present results. Dilutions of sera however can be readily varied, together with relevant specificity controls, to generate antibody titers from the COVID-19 IB assays.
The criteria for antibody positivity utilized the necessary recognition of at least two of the four SARS-CoV-2 proteins for optimizing the specificity of COVID-19 IgG and IgM IB assays. The RBD lies within the S1 region of the S protein but the detection of RBD by antibodies did not parallel the detection of S1, with RBD being detected by fewer sera and variably at different time periods compared to S1. Epitopes in regions other than the RBD in S1 are therefore importantly antigenic in patients. Antibodies to the RBD in particular and the more N terminal region of S1 are important for neutralizing virus infectivity by preventing binding to host cells [
14,
15]. Some antibodies to S2 may also neutralize infectivity by inhibiting cell fusion and virus entry [
16,
17]. Measurement of antibody titers in the IB assay may be relevant as IgG antibody titers to the S protein measured by ELISA correlate with virus-neutralizing antibody titers in persons vaccinated with S [
8], although this correlation is weaker in non-hospitalized patients [
18]. Other data suggest that antibody levels to RBD and other viral antigens are higher in more severely ill hospitalized patients [
19,
20], which may be consistent with the weaker anti-RBD antibody responses in non-hospitalized patients seen in the present study.
One of the 37 patients did not show detectable antibodies when tested on days 15, 30 and 44, a phenomenon which has previously been observed in other non-hospitalized patients [
18]. It is possible that this patient’s sera might have possessed detectable levels of antibodies had it been tested prior to day 10 or between days 51 to 65, periods after infection when the overall sensitivity of detecting antibodies was 100% in the COVID-19 IB assays. The decline in antibodies in the 100 to 154 day period after a positive RT-qPCR test may be partly characteristic of the relatively mild disease studied here since antibody levels are reported to be more sustained in severely ill patients [
21]. The findings emphasize the importance of detecting both IgG and IgM antibodies rather than either antibody class alone, and at different times after infection, for assessing seroconversion in COVID-19, which is consistent with the findings in symptomatic patients from China [
20].
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