Immunology

Advanced immunoassays are crucial in assessing antibody responses, serving immune surveillance goals, characterising immunological responses to evolving viral variants, and guiding subsequent vaccination initiatives. This protocol outlines an indirect ELISA protocol to detect and quantify virus-specific antibodies in plasma or serum after exposure to viral antigens. The assay enables the measurement of IgG, IgA, and IgM antibodies specific to the virus of interest, providing qualitative and quantitative optical densities and concentration data. Although this protocol refers to SARS-CoV-2, its methodology is versatile and can be modified to assess antibody responses for various viral infections and to evaluate vaccine trial outcomes.

Key features

• This protocol builds upon previously described methodology [1] explicitly tailored for SARS-CoV-2 and broadens its applicability to other viral infections.

• The protocol outlines establishing antibody responses to SARS-CoV-2 infections by determining optical densities and concentrations from blood plasma or serum.

Graphical overview

Summary of the conventional ELISA (A) vs. sensitive ELISA (B) procedures. In both A and B, wells are coated with a capture antigen, such as the spike protein, while in (C) they are coated with human Kappa and Lambda capture antibodies. For the conventional ELISA (A), wells with immobilised capture antigens receive serum/plasma containing the target antibody (A1 and B1). This is followed by an HRP-conjugated detection antibody specific to the captured antibody (A2 and B2) and then a substrate solution that reacts with the HRP, producing a colour proportional to the concentration of the antibody in the serum/plasma (A3 and B3). The reaction is halted, and absorbance is measured. In the sensitive ELISA (B), after the serum/plasma addition (A1 and B1), a Biotin-conjugated primary detection antibody is introduced (A2 and B2). Depending on the target antibody, a secondary streptavidin-HRP conjugated detection antibody is added for IgG or IgM (3a) or a poly-HRP 40 detection antibody for IgA (3b). A substrate is introduced, producing a colour change proportional to the antibody concentration (A4 and B4). The reaction is then stopped, and absorbance is measured. In Panel C, wells are coated with human Kappa and Lambda capture antibodies. Serial dilutions of a known antibody standard are introduced. After undergoing the standard ELISA steps, a detection antibody is added, specifically binding to the Ig standard antibody. Subsequently, a substrate solution causes a colour change proportional to the antibody concentration in the serum/plasma. The reaction is halted, and the absorbance of each well is measured. The resulting optical densities from the coated wells form the standard curve, plotting the absorbance against concentrations.

Platelets play an important role in hemostasis by forming clots and stopping bleeding. In immune thrombotic conditions, platelets and leukocytes are aberrantly activated by pathogenic antibodies resulting in platelet aggregates and NETosis, leading to thrombosis and thrombocytopenia. A simple assay that assesses platelet function and antibody activity is light transmission aggregometry. This assay can be used to determine antibody activity in patients with disorders such as heparin-induced thrombocytopenia (HIT) and vaccine-induced thrombotic thrombocytopenia (VITT). Briefly, for detection of pathogenic antibody, platelet-rich plasma (PRP) is treated with a specific agent (e.g., patient sera or purified patient antibodies) with constant stirring. Upon activation, platelets undergo a shape change and adhere to each other forming aggregates. This causes a reduction in opacity allowing more light to pass through PRP. Light transmission through the cuvette is proportional to the degree of platelet aggregation and is measured by the photocell over time. The advantage of this protocol is that it is a simple, reliable assay that can be applied to assess antibody activity in thrombotic conditions. Light transmission aggregometry does not require the use of radioactive reagents and is technically less demanding compared with ¹⁴C-serotonin release assay, another common assay for detecting antibody activity.

Key features

• This protocol can be used to assess platelet function and to detect platelet activating antibodies in diseases such as HIT and VITT.

• Does not require radioactive reagents, requires an aggregometer; based on the light transmission aggregometry protocol, adapted for detection of VITT and other platelet-activating antibodies.

• Two positive controls are required for reliable detection of antibodies in diseases such as HIT/VITT, namely a weak HIT/VITT antibody and a physiological agonist.

• For detection of HIT/VITT antibodies, it is essential to use donors known to have platelets reactive to these antibodies to avoid false negative results.

Immunoglobulins are proteins produced by the immune system, which bind specifically to the antigen that induced their formation and target it for destruction. Highly purified human immunoglobulins are commonly used in research laboratories for several applications, such as in vitro to obtain hybridomas and in vivo animal immunisation. Several affinity purification methods are used to purify immunoglobulins from human serum, such as protein A/G Sepharose beads, polyethylene glycol, and caprylic acid ammonium sulphate precipitation. Here, we provide a detailed protocol for purification of high-quality IgG from human serum, using affinity chromatography with protein G. The protocol is divided into four main steps (column preparation, serum running, wash, and elution) for IgG purification, and two extra steps (protein dialysis and sucrose concentration) that should be performed when buffer exchange and protein concentration are required. Several IgG affinity purification methods using protein A or G are available in the literature, but protein A has a higher affinity for rabbit, pig, dog, and cat IgG, while protein G has a higher affinity for mouse and human IgG. This affinity-based purification protocol uses protein G for a highly specific purification of human IgG for animal immunization, and it is particularly useful to purify large amounts of human IgG.

Graphical abstract

IgG purification protocol. The IgG purification protocol consists of four main steps (column preparation, serum running, wash, and elution) and two extra steps (protein dialysis and concentration). a. Diluted serum is added to the protein G beads and IgG binds to the Fc receptors on protein G beads. b. Beads are washed in Hartman’s solution to fully remove the complex protein mixture (multicolour shapes, as depicted in the graphical abstract). c. IgG (orange triangles, as depicted in the graphical abstract) are removed from protein G with glycine and collected in Tris buffer. d. The IgG is transferred into a semi-permeable membrane (‘snake skin’) and allowed to dialyse overnight for buffer exchange with a physiological solution (Hartmann’s).

Neutralizing antibodies (NAbs) are of particular importance because they can prevent binding of the receptor binding domain (RBD) of the spike protein (S protein) to the angiotensin-converting enzyme 2 (ACE2) receptor present at the surface of human cells, preventing virus entry into the host cells. The gold standard method for detection of NAbs is the plaque reduction neutralization test (PRNT). Based on the measurement of cell lysis due to viral infection, this test is able to detect antibodies that prevent cell infection (Muruato et al., 2020; Lau et al., 2021). This technique requires the use of live pathogens, i.e., SARS-CoV-2 in this case, and must be done in a biosafety level 3 (BL3) laboratory. In addition, it requires expensive installations, skillful and meticulous staff, and a high workload, which prevents its wide implementation even in research laboratories. A SARS-CoV-2 pseudovirus will express the S protein responsible for cell entrance, but will not express the pathogenic genetic material of the virus, making them less dangerous for laboratory staff and the environment.

Graphic abstract:

The SARS-CoV-2 pandemic and vaccination campaign has illustrated the need for high throughput serological assays to quantitatively measure antibody levels. Here, we present a protocol for a high-throughput colorimetric ELISA assay to detect IgG antibodies against the SARS-CoV-2 spike protein. The assay robustly distinguishes positive from negative samples, while controlling for potential non-specific binding from serum samples. To further eliminate background contributions, we demonstrate a computational pipeline for fitting ELISA titration curves, that produces an extremely sensitive antibody signal metric for quantitative comparisons across samples and time.

For enveloped viruses, such as SARS-CoV-2, transmission relies on the binding of viral glycoproteins to cellular receptors. Conventionally, this process is recapitulated in the lab by infection of cells with isolated live virus. However, such studies can be restricted due to the availability of high quantities of replication-competent virus, biosafety precautions and associated trained staff. Here, we present a protocol based on pseudotyping to produce recombinant replication-defective lentiviruses bearing the SARS-CoV or SARS-CoV-2 attachment Spike glycoprotein, allowing the investigation of viral entry in a lower-containment facility. Pseudoparticles are produced by cells transiently transfected with plasmids encoding retroviral RNA packaging signals and Gag-Pol proteins, for the reconstitution of lentiviral particles, and a plasmid coding for the viral attachment protein of interest. This approach allows the investigation of different aspects of viral entry, such as the identification of receptor tropism, the prediction of virus host range, and zoonotic transmission potential, as well as the characterisation of antibodies (sera or monoclonal antibodies) and pharmacological inhibitors that can block entry.

Graphic abstract:

SARS-CoV and SARS-CoV-2 pseudoparticle generation and applications.

The recombinant receptor-binding domain (RBD) of the viral spike protein from SARS-CoV-1 and 2 are reliable antigens for detecting viral-specific antibodies in humans. We and others have shown that the levels of RBD-binding antibodies and SARS-CoV-2 neutralizing antibodies in patients are correlated. Here, we report the expression and purification of properly folded RBD proteins from SARS and common-cold HCoVs in mammalian cells. RBD proteins were produced with cleavable tags for affinity purification from the cell culture medium and to support multiple immunoassay platforms and drug discovery efforts.

Graphic abstract:

High-Yield Production of Viral Spike RBDs for Diagnostics and Drug Discovery