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  • April Wong

SPR to Examine Cross-Reactivity of Antibodies with Native and Variant SARS-CoV-2 Spike Proteins

Updated: Oct 2


The rise of SARS-CoV-2 variants of concern (VOCs) adds more challenges to the public health system and delays the return to a near pre-pandemic life and economy. Therefore, a thorough understanding of how protective antibodies are produced from the native strain (i.e. the strain initially from Wuhan, China) against VOCs would be beneficial in outlining public health guidelines and vaccination programs.

SARS-CoV-2 invades cells in the human respiratory tract by membrane fusion. First, the receptor-binding domain (RBD) from a subunit of the spike proteins present on the virus binds to the angiotensin-converting enzyme 2 (ACE-2) receptor found on the cell membranes in the respiratory tract (see Fig. 1). However, neutralizing antibodies (from natural infection or vaccine) prevents the viruses from binding to the ACE-2 receptors on the cell surfaces.

For VOCs, there are mutations in the spike protein that may lead to a lowered neutralization antibody response for those individuals who have been infected with the native coronavirus strain. The same scenario can apply to those who have been immunized, as the current mRNA and viral vector-based vaccines were produced according to the genetic makeup and structure of the native strain.

Therefore, it is of utmost importance to investigate the ability of these antibodies to bind to the spike proteins of VOCs to prevent infection. Recently, a research team, led by Joelle Pelletier and Jean-François Masson from Université de Montréal, has investigated the cross-reactivity of antibodies from COVID-19 positive individuals (non-hospitalized) against the native and beta variant (B.1.351) SARS-CoV-2 spike proteins [1].

Fig. 1. The simplified scheme demonstrates how neutralizing antibodies against the spike proteins of SARS-CoV-2 help block the virus from infecting cells in the respiratory tract.

ELISAs and cell-based neutralization assays are typically used to study the humoral response against SARS-CoV-2. However, these assays are lengthy and expensive, and cell-based neutralization assays require the use of live viruses in biosafety level 3 (BSL3) labs. Therefore, surface plasmon resonance (SPR) is becoming an essential technique in this field because it can complement biochemical data from ELISA and be a surrogate for cell-based neutralization assays.

This blog intends to highlight the researchers' findings and emphasize how Affinité Instruments' compact P4SPR can provide information on the cross-reactivities of antibodies against native and variant spike proteins. Furthermore, the P4SPR offers an alternative solution to cell-based neutralization assay that is less costly, more rapid, and safer.

Experimental Details

The following is a summary of the experiments that were carried out in the study [1].

1. Detection of Anti-Spike Antibodies by SPR

Briefly, the SPR experiment procedures used to determine the level of IgG antibody binding to the native or beta strain spike protein are similar to Steps 1-2 in Fig. 2 by using a quad inlet P4SPR. The final detection step involved adding anti-human IgG as a secondary antibody in a sandwich assay, similar to the one described in the HER2 Biomarker blog.

2. Dissociation Constants of Antibody-Spike Protein Interactions by SPR

The KDs were determined by constructing a calibration curve using a dual inlet P4SPR such that triplicate measurements can be done for each dilution. First, the spike protein from the native or beta variant was immobilized on the SPR chips as in Step 1 in Fig. 2. Then, decreasing dilutions of the sera containing the antibodies were injected into the P4SPR. Finally, the signals at equilibrium were fit into a 1:1 Langmuir model to obtain the KD value for either the native or beta variant spike protein and antibody interaction.

3. SPR used as a Pseudo-Neutralization Assay

SPR was used as a virus-free, in vitro method to replace cell-based neutralization assay. In a nutshell, spike proteins from the native or beta variant were immobilized on an Affinité Instruments gold sensor chip (Step 1, Fig. 2) followed by the injection of COVID-19 positive or negative sera (Step 2, Fig. 2). The ability of the antibodies to bind to the spike proteins in positive sera was compared to negative sera by observing the difference in SPR signals obtained after the addition of recombinant ACE-2 (Step 3, Fig. 2). The presence of antibodies in COVID-19 positive sera should bind to the immobilized spike proteins, inhibiting the ACE-2 receptors from binding to the spike proteins. After the injection of ACE-2, the change in SPR response should be lower for COVID-19 positive sera compared to negative sera. These measurements were then compared to the positive and negative controls (for background correction), set up on an identical chip in a quad inlet P4SPR.

1. Spike protein (native or beta variant) was immobilized onto the SPR sensor chip.

2. Serum from COVID-19 positive individuals were injected into the P4SPR containing the sensor chip from Step 1the antibodies in the serum bound to the spike proteins.

3. Recombinant human ACE-2 receptors were injected and left to react with any remaining spike proteins.

Fig. 2. The steps (1-3) that were used in the SPR-based pseudo-neutralization assay.

Major Discoveries

According to both ELISA and SPR results, the antibodies from COVID-19 positive individuals bound to the spike proteins of both native and beta (B.1351) strains [1]. However, the signals were lower for the beta spike protein [1]. Next, dissociation constants (KD) between the anti-spike antibodies and native or beta spike proteins were determined. For both interactions, the KDs were higher for antibodies analyzed at 4 weeks than those at 16 weeks [1]. This means that the antibodies' affinity towards the spike proteins starts to decline sometime between 4 to 16 weeks [1]. Nevertheless, these results proved that sera of individuals who were never exposed to the beta strain contained anti-spike antibodies that cross-reacted with the beta strain spike protein up to 16 weeks post-diagnosis.

The second part of this study involved using SPR as an in vitro strategy to observe any inhibition of any spike protein-ACE-2 receptor interactions by anti-spike antibodies in sera of COVID-19 positive individuals (Fig. 2). The antibodies did inhibit the interaction, albeit lower for the beta spike protein, because those antibodies were from individuals exposed to the native strain of the SARS-CoV-2 virus [1].

Key Advantages of the P4SPR Highlighted from this Study

Not only has this study demonstrated that antibodies produced from the native SARS-CoV-2 can cross-react with the spike protein from the beta variant, but it has also revealed some advantages of using the P4SPR compared to ELISA. For example, the dilution factor required for SPR assays was at 1:5 compared to 1:50 for an antigen-down colourimetric ELISA. Other SPR experiments in the study did not even require any dilutions. Furthermore, for SPR assays, both high and low concentrations or high and low-affinity interactions can be observed, versus only high affinity and high concentration of antibodies for ELISA.

Lastly, SPR can be used to perform a pseudo-neutralization study, negating the need to use live viruses in a cell-based neutralization assay in a BSL3 lab. It is less expensive, takes less time, and the procedure is certainly not as complicated. Most importantly, SPR has proven to generate results comparable to ELISA while exhibiting the same advantages as mentioned above [2].


This study, assisted by Affinité's P4SPR system, revealed important information about the cross-reactivity of anti-spike antibodies from naturally infected individuals of the original SARS-CoV-2 strain.

First, the antibodies were able to bind to the spike proteins of the beta variant, albeit at a lower level than the native spike proteins. Second, they were able to inhibit the interactions between ACE-2 and the spike protein. Third, the anti-spike antibodies' affinity towards the spike protein is highest for both native and beta strain shortly after infection and decreases afterwards. Furthermore, the SPR results from this study correlated with ELISA, which agreed with a previous paper. Lastly, the P4SPR was able to quantify the inhibition level of the spike protein-ACE-2 receptor interaction by anti-spike antibodies without performing a complex cell-based neutralization assay in a BSL-3 lab.

The Affinité Advantage

Affinité Instruments’ P4SPR™ is a very user-friendly instrument that can be tailored to accommodate all types of surface chemistries. In addition, samples do not need much preparation and can be directly injected into the device. The P4SPR™, compared to a traditional immunoassay such as ELISA, provides fast, real-time affinity and or kinetic data.

Simplicity - Fast training, fast results

Versatility - Pharmaceutical, biosensing, assay development applications

Economy - Affordable, accessible

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[1] Cross-reactivity of antibodies from non-hospitalized COVID-19 positive individuals against the native and B.1.351 SARS-CoV-2 spike proteins | Research Square.

[2] Cross-validation of ELISA and a portable surface plasmon resonance instrument for IgG antibody serology with SARS-CoV-2 positive individuals - Analyst (RSC Publishing)