Surface Plasmon Resonance Immunosensor for Direct Detection of Antibodies against SARS-CoV-2 Nucleocapsid Protein

Abstract

The strong immunogenicity of the SARS-CoV-2 nucleocapsid protein is widely recognized, and the detection of specific antibodies is critical for COVID-19 diagnostics in patients. This research proposed direct, label-free, and sensitive detection of antibodies against the SARS-CoV-2 nucleocapsid protein (anti-SCoV2-rN). Recombinant SARS-CoV-2 nucleocapsid protein (SCoV2-rN) was immobilized by carbodiimide chemistry on an SPR sensor chip coated with a self-assembled monolayer of 11-mercaptoundecanoic acid. When immobilized under optimal conditions, a SCoV2-rN surface mass concentration of 3.61 ± 0.52 ng/mm² was achieved, maximizing the effectiveness of the immunosensor for the anti-SCoV2-rN determination. The calculated K_D value of 6.49 × 10^-8 ± 5.3 × 10^-9 M confirmed the good affinity of the used monoclonal anti-SCoV2-rN antibodies. The linear range of the developed immunosensor was from 0.5 to 50 nM of anti-SCoV2-rN, where the limit of detection and the limit of quantification values were 0.057 and 0.19 nM, respectively. The immunosensor exhibited good reproducibility and specificity. In addition, the developed immunosensor is suitable for multiple anti-SCoV2-rN antibody detections.

Keywords: SARS-CoV-2 nucleocapsid protein; antibody; immunosensor; surface plasmon resonance.

Figure 1

Simplified schematic illustration of SPR immunosensor fabrication and direct anti-SCoV2-rN detection. (1) Formation of MUA SAM on the surface of the SPR sensor chip. (2) Covalent immobilization of SCoV2-rN using carbodiimide conjugation chemistry. (3) Interaction of immobilized SCoV2-rN with specific antibodies—anti-SCoV2-rN.

Figure 2

Dependence of Au/SCoV2-rN/anti-SCoV2-rN complex dissociation and surface regeneration efficiency on the (A) nature of the regeneration solution and (B) regeneration duration using 10 mM NaOH and 0.5% SDS solution. Conditions: 500 nM SCoV2-rN; 10 nM anti-SCoV2-rN. Error bars represent the standard deviation of three measurements (n = 3).

Figure 3

Effect of the pH value of acetate buffer used for SCoV2-rN dilution on the magnitude of the response of SCoV2-rN and anti-SCoV2-rN immune complex formation. Conditions: 500 nM SCoV2-rN; immobilization duration—1200 s; 15 nM anti-SCoV2-rN; 50 mM acetate buffers with different pH. Error bars represent the standard deviation of three measurements (n = 3).

Figure 4

Effect of initial SCoV2-rN concentration on the magnitude of the response to SCoV2-rN immobilization and interaction with anti-SCoV2-rN. Conditions: SCoV2-rN was diluted in 50 mM acetate buffer, pH 5.3; 15 nM anti-SCoV2-rN. Error bars represent the standard deviation of three measurements (n = 3).

Figure 5

SPR kinetic study of immune complex formation between immobilized SCoV2-rN protein and anti-SCoV2-rN present in the buffer. Conditions: 500 nM SCoV2-rN; interaction time—1500 s; 1, 3, 5, 10, and 30 nM anti-SCoV2-rN.

Figure 6

(A) SPR sensograms recorded during the analysis of solutions with different anti-SCoV2-rN concentrations using a direct immunoassay format and (B) a calibration curve. (C) Dependence of SPR angle shift on anti-SCoV2-rN concentration. Conditions: 500 nM SCoV2-rN; interaction time—600 s; 0.3, 0.5, 1, 3, 5, 10, 15, 20, 30, 40, and 50 nM of anti-SCoV2-rN. Error bars represent the standard deviation of three measurements (n = 3).

Figure 7

Non-specific interaction study. The SPR signal registered during (1) non-specific binding of anti-COMP antibodies on the SCoV2-rN-modified SPR sensor chip surface, and (2) formation of the immune complex of immobilized SCoV2-rN and anti-SCoV2-rN antibodies present in the sample. The study was performed by injecting 50 nM of anti-COMP or anti-SCoV2-rN in 10 mM PBS solution, pH 7.4.