Enzyme-Linked Immunosorbent Assay: An Adaptable Methodology to Study SARS-CoV-2 Humoral and Cellular Immune Responses

Abstract

The Enzyme-Linked Immunosorbent Assay is a versatile technique, which can be used for several applications. It has enormously contributed to the study of infectious diseases. This review highlights how this methodology supported the science conducted in COVID-19 pandemics, allowing scientists to better understand the immune response against SARS-CoV-2. ELISA can be modified to assess the functionality of antibodies, as avidity and neutralization, respectively by the standardization of avidity-ELISA and surrogate-neutralization methods. Cellular immunity can also be studied using this assay. Products secreted by cells, like proteins and cytokines, can be studied by ELISA or its derivative Enzyme-linked immunospot (ELISpot) assay. ELISA and ELISA-based methods aided the area of immunology against infectious diseases and is still relevant, for example, as a promising approach to study the differences between natural and vaccine-induced immune responses against SARS-CoV-2.

Keywords: ELISA; SARS-CoV-2; antibody; cytokine; immune response.

Figure 1

(A) SARS-CoV-2 structural antigens, used for immunoassays. Nucleocapsid and Spike proteins are the most abundant antigens, therefore, they are used for improved sensitivity. Antigen-subunits, as S1 and S2, or RBD and NTD, can provide more specific results. (B) The expected IgM/IgG curves following exposure to antigen through natural infection or vaccination. The maturation of immune response usually leads to initial IgM induction and, while titers of this antibody class decrease, IgG titers increase and persist for longer periods, which varies according to the pathogen considered. Below it, kinetics of SARS-CoV-2 antigens and antibodies detection is represented. Differently from the expected, titers of IgA, IgM, and IgG antibodies increase in parallel. The time to achieve seronegativity and the particularities of vaccine-induced humoral response are still being elucidated (figure created with BioRender).

Figure 2

Differences in non-functional and functional enzyme-linked assays. While non-functional assays (A), like regular ELISA, detect antibodies that are bound with the antigen, functional assays detect antibodies regarding specific characteristics. (B) Avidity assays consist in adding a chaotropic agent that will disturb the epitope-paratope interaction, disrupting weak bindings. Only avid antibodies keep attached and are revealed in the final result. Both A and B assays should consider which antigen of SARS-CoV-2 was used to coat the plate (e.g., RBD, Spike protein, Nucleocapsid protein) and the specificity of the anti-Ig used for detection (e.g., anti-IgA, anti-IgM, anti-IgG). For quantitative results, the higher the signal, the higher the antibody quantity. (C) Surrogate-virus neutralization assays, based on a competitive ELISA, allow the interaction between labelled-RBD and the sample, after that, the mix is incubated in ACE-2 coated plates. Therefore, free-RBD binds to the plate and complexes antibody-labelled-RBD, which cannot bind with it, are washed. It should be noted that the assay considers all anti-RBD antibodies, despite its Ig class. For quantitative results, the lower the signal, the higher is the antibody quantity. (figure created with BioRender).

Figure 3

Immune cells and SARS-CoV-2 infection. SARS-CoV-2 is internalized after the RBD-ACE2 interaction. Natural-killer (NK) cells can identify infected cells and kill them after the release of cytotoxic proteins (like perforin and granzyme). Dendritic cells (DC) are responsible for capture, processing, and presentation of pathogen to lymphocytes, to assemble the adaptive immune response. After activation and antigen presentation, plasma (B) cells produce and release antibodies that, for example, can neutralize the virus by inhibition of RBD-ACE2 interaction; while cytotoxic T cells (CD8+ cell) recognize infected cells and kill it by releasing cytotoxic proteins. Auxiliary T cells (CD4+ cell) differentiate to certain T helper (Th) types, releasing cytokines that modulate the immune environment, supporting different arms of response. A well-orchestrated immune response is necessary to control the infection without harming the host and the products secreted by cells (e.g., proteins, cytokines, immunoglobulins) can be enzyme-linked detected. (figure created with BioRender).