Memory B cells and/or circulating plasma cells can be identified by their capability to secrete antigen-specific antibodies long after first encountering an antigen [1
]; thus, they can provide important information related to past exposure. This feature has been employed to setup in vitro assays to detect and count antibody-secreting B cells through ELISPOT platforms [2
]. With this method, antibody-producing cells secrete antibodies that bind to an antigen-coated plastic surface. The antigen–antibody complex is subsequently detected by a secondary antibody or substances covalently linked to enzyme/fluorochromes, and it is visualized as spots and can be counted using colorimetric or fluorescent methods [3
]. ELISPOT has been successfully applied in vitro to measure the number of antibody-producing B cells, employing immobilized antigens from various pathogens including viral antigens [4
]. Direct measurement of memory B-cells in peripheral blood can establish the magnitude and longevity of protection against infections and may be used in different fields of biomedical research, including epidemiology and vaccine development. We previously modified ELISPOT into a cell-based ELISA (cell-ELISA) that does not require the spot counting step, since the leukocytes are removed after incubation with the immobilized antigen, and the antigen-bound specific IgGs secreted in vitro by plasma cells are assayed by immunoenzymatic analysis [6
]. Of note, quantification of B-cell secreted antibodies, if applicable in the clinical context, might be important for recognizing subjects who have encountered pathogens and identifying the occurrence of a disease. The latter is especially crucial during a pandemic, as in the case of the ongoing outbreak caused by the novel betacoronavirus named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), first isolated in China in December 2019 and responsible for a highly transmissible disease named COVID-19. It is generally more severe in older age and is characterized by a range of mild respiratory symptoms that may advance to acute respiratory distress syndrome (ARDS) and multi-organ dysfunction [7
]. Following host cell infection, SARS-CoV-2 triggers both innate and specific immune responses, but the precise immune pathways involved in activating the antivirus host defense mechanisms remain to be elucidated. To verify whether infection is ongoing, viral presence is diagnosed by analyzing the expression of viral genes via real-time PCR in biological material collected through nasopharyngeal swabs. The immune response can instead be measured by screening for the presence of specific antibodies in the blood using ELISA serological analyses [8
]. The coronavirus envelope spike S1 protein is responsible for receptor binding and fusion to host cells, and it is involved in tropism and transmission capability [9
]. The S1 protein has been shown to be a serological marker for COVID-19 [10
]. Consequently, serological analyses via ELISA evaluate the presence of specific IgM/IgG by employing the recombinant spike protein S1 as adsorbed antigen. Antibody titers can be measured in intermediate and later stages of the infection (from day 7–10 onward) and are indicative of an antibody response [11
]. The importance and robustness of ELISA systems in monitoring serum antibody response to the COVID-19 virus are confirmed by the number of diagnostic kit brands that are present on the market. However, a major limitation of ELISA-based platforms is circulating antibodies may become less and less detectable over time following vaccination or infection. On the other hand, circulating antigen-specific memory B cells can be present and persist long after immunization [4
]. Upon reactivation, memory B cells are able to mount an antibody response decades after first encountering an antigen; hence, providing critical information regarding previous pathogen exposure. This feature is exploited by monitoring the humoral response of B cells, i.e., their capability to secrete in vitro specific IgG antibodies when restimulated with immunization antigen(s) [3
A cell-based ELISA test adapted for COVID-19 would be useful to measure IgG specific for the S1 protein of SARS-CoV-2 produced in vitro by B cells, even when circulating antibodies become less detectable over time. Furthermore, by combining this assay with ELISA, it is possible to simultaneously detect the level of S1-specific plasma IgG and the presence of IgG-producing B cells in peripheral blood mononuclear cells (PBMCs) in plasma from the same donor. Such an approach may be helpful to improve serological analysis and determine the immunization status for SARS-CoV-2.
In this brief communication, we describe a new cell-ELISA, performed in combination with a commercially available COVID-19 serological test, in a cohort of 45 subjects. This assay is used to detect in vitro cell-produced IgG directed against the SARS-CoV-2 S1 protein. Although further work is required to confirm our preliminary results, we propose this cell-ELISA as a potential supplementary diagnostic tool useful for ruling out individuals who have very low serum IgG levels undetectable by ELISA, and thus improving the assessment of past infection and immune response in subjects during the COVID-19 pandemic.
Accurate tests able to identify the SARS-CoV-2 virus and monitor the presence of antiviral antibodies are key in detecting those who have had an immune response to the virus, to help the management and surveillance of the virus, and thus to fight the COVID-19 pandemic. Diagnosis for SARS-CoV-2 is currently performed after running qRT-PCR of selected viral genes from nasal/respiratory tract swabs, monitoring the immune humoral response by serological assays, and/or measuring circulating IgM/IgG antibodies using various immunoenzymatic ELISA platforms available on the market. Previous studies have reported that serological testing may be useful to identify asymptomatic or subclinical infection with SARS-CoV-2 among medical workers in close contact with patients with COVID-19 [12
]. However, in another study conducted on healthcare workers at risk for SARS-CoV-2 transmission, the majority of participants with positive IgG had a significant decline in antibody levels after one month [13
]. In this context, information on the presence of memory B cells can be pivotal, since some studies have shown that circulating antibodies can shift to undetectable levels after vaccination or infection, while circulating antigen-specific memory B cells are still present [4
]. In order to acquire information on the mounting of a B cell memory response for SARS-CoV-2, in this study, we adapted a simplified ELISPOT platform [5
] to detect in vitro produced IgG for the spike S1 protein of SARS-CoV-2, which has already shown to be antigenic in humans and has been employed as an antigen in serological ELISAs [7
]. The principle of this cell-ELISA is based on the use of PBMCs cultured in plastic wells pre-coated with recombinant S1 protein, as supplied in commercial ELISA kits. The cell-ELISA platform was tested with PBMCs obtained from donors whose plasma was already probed by ELISA 35–60 days earlier (ELISA 1). This timing was selected, taking into consideration recent data on the production of neutralizing antibodies at 39 days after infection [16
], to discriminate between the reported half-life of IgG, being less than 30 days [18
], and the presence of memory B cells and plasma cells, detected as early as 7 days after primary immunization [19
], even in the absence of stimulatory factors (such as R848 and IL-2), as already reported for viral antigens [5
Cell-ELISA positivity was assessed, using the arbitrary threshold of an OD450 value of 0.065 (as described above), on data obtained with PBMCs incubated with CXM, on spontaneous releases and from PBMCs cryoconserved before the COVID-19 pandemic.
Cell-ELISA data obtained for all tested samples, reported in Figure 1
, showed 29 negatives and 16 positives. Data were reported in an age-dependent manner, where those tested positive by cell-ELISA (black dots) and negative by serological ELISA 2 (yellow dots) were indicated. Importantly, only one of the positive samples by ELISA 1 and ELISA 2 tested negative (at borderline) by cell-ELISA, and thus confirming the robustness of the assay. To our knowledge, this is the first description of an assay measuring IgG produced in vitro by B cells and specific for the SARS-CoV-2 S1 spike protein, an antigen shown to induce specific anti-COVID-19 IgG [10
Although no conclusive statements can be drawn because of the relatively small size of the analyzed population, five individuals out of 45 (11.2%) tested negative by both ELISA1 and ELISA2 but positive by cell-ELISA, and thus, they appear to have circulating B cells that produce antibodies against SARS-CoV-2, likely at levels that are undetectable in the serum. This challenges the negative results from serological screening and suggests that the cell-ELISA may detect S1-specific IgG secreted by B cells even when a serological ELISA does not. This assumption is reinforced by the observation that all 16 subjects tested positive by cell-ELISA, including those who ELISA tested as negative and claimed to have had symptoms attributable to COVID-19.
This finding confirms previous observations describing that circulating antigen-specific memory B cells can be present and persist after immunization, even in the absence of detectable serum IgG [4
]. In addition, although preliminary, our results further extend and improve recent findings on the kinetics of serum antibody responses in COVID-19 patients, where the ELISA for IgM/IgG was claimed to detect 75% of SARS-CoV-2-infected patients in the first week [20
To confirm effective IgG production by B cells, in vitro cultures of PBMC were also evaluated in the presence of CXM, an antibiotic drug known to block initial phases of protein elongation in ribosomes, which have already been employed in a cell-ELISA animal model [5
]. The data reported in Figure 2
suggest effective inhibition of protein synthesis and secretion by CXM and likely the blocking of IgG production by plasma cells. The same data also suggest the need to use a minimum concentration of 9 × 106
/mL PBMCs to gain reliable cell-ELISA data.
Even though the results we obtained by cell-ELISA appeared specific, as demonstrated by the CXM control, they were mainly based on low OD450 values. This probably is due to the small number of antibody-secreting cells present in peripheral blood and/or to the low sensitivity of reagents in the kit, which are calibrated for plasma/serum IgG. However, we choose to use a commercial kit to measure cell-secreted antiviral antibodies in order to support the enforceability of the assay in standard diagnostic labs. In addition, it is possible to select the antibody class to be tested by the secondary antibody employed.
To understand the possible levels of IgG secreted by PBMCs and the sensitivity threshold of the assay, the OD450 values of positive samples were compared to those from reference concentrations of human γ-globulin determined by ELISA. Obtained data suggest a minimal assay sensitivity of 7 ng IgG/well, which increased up to 50 ng/well of secreted IgG in donors. Interestingly, the donor whose PBMCs secreted the highest level of anti-S1 IgG in vitro tested negative in both serological ELISA 1 and ELISA 2 and declared no symptoms.
In conclusion, we propose here a modified ELISPOT platform, which was converted to a simpler cell-ELISA adapted to detect the S1 spike protein of SARS-CoV-2 and able to monitor the presence of memory B and plasma cells in the blood.
The proposed cell-ELISA, although needing more work addressed at increasing the number of donors and sampling over time, may improve upon conventional plasma or serum-based ELISAs, especially in the case of undetectable IgGs in serological analysis. Importantly, the assay could be of help in assessing the immunization status via S1 protein detection, and thus, providing a clearer picture of infection progression, and can readily be applied in clinical trials. Such important information may prove crucial in establishing seroprevalence in a more accurate way, in determining which individuals will benefit more than others from vaccination, and eventually in defining the efficacy of new vaccines.