The effect of heat inactivation of ferrets serum samples on the detection of SARS-CoV-2-specific IgG antibodies in elisa

Abstract

BACKGROUND: Determination of serum antibody levels to the novel coronavirus SARS-CoV-2 is a necessary tool for assessing humoral immunity in COVID-19 patients or individuals vaccinated with specific vaccines, as well as for studying the immune responses to the viral antigens in animal models. Serum specimens of infected humans and animals are considered potentially infectious material, and therefore heat inactivation of samples at 56 °C for 1 hour is recommended to reduce the risk of infection of personnel during serological studies. However, this procedure may affect the detection of virus-specific IgG and IgM antibodies, making interpretation of the results difficult.

AIM: The goal is to evaluate the effect of heat inactivation of ferret serum samples on the binding of IgG antibodies to the SARS-CoV-2 antigens.

MATERIALS AND METHODS: Serum samples of SARS-CoV-2 naive and immune ferrets were analyzed in three variants: (1) native sera, (2) serum samples than were heated at 56°C for 1 hour, and (3) serum samples that were treated with receptor-degrading enzyme (RDE). The samples were studied in enzyme-linked immunosorbent assay (ELISA) using recombinant RBD protein (receptor-binding domain of SARS-CoV-2 S-protein) as a substrate, followed by determination of specific antibody levels before and after treatments.

RESULTS: It has been shown that heat inactivation of the ferrets’ naive serum samples can lead to false-positive results, while RDE treatment can neutralize the effect of non-specific binding of IgG antibodies to the RBD domain of the SARS-CoV-2 S protein.

CONCLUSIONS: Structural rearrangement of SARS-CoV-2-specific IgG antibodies and the formation of immunoglobulin complexes during heat inactivation of serum samples can affect the avidity of the antigen-antibody complex and lead to false-positive results when performing enzyme immunoassay. One of the possible methods to reduce the risk of “artifacts” is the treatment of blood sera with RDE, which eliminates the effect of heat inactivation.

Keywords

SARS-CoV-2 / enzyme immunoassay / heat inactivation / blood sera / receptor-binding domain / recombinant protein

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