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 56C 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.

Assessing the levels of serum IgG antibodies is widely used to measure immunity to influenza and the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) after natural infection or vaccination with specific vaccines, as well as to study immune responses to these viruses in animal models. For safety reasons, sometimes serum specimens collected from infected individuals are subjected to heat inactivation at 56 °C to reduce the risk of infecting personnel during serological studies. However, this procedure may affect the level of virus-specific antibodies, making the results of antibody immunoassays uninterpretable. Here, we evaluated the effect of the heat inactivation of human, ferret and hamster serum samples on the binding of IgG antibodies to the influenza and SARS-CoV-2 antigens. For this, serum samples of naive and immune hosts were analyzed in three variants: (i) untreated sera, (ii) heated at 56 °C for 1 h, and (iii) treated with receptor-destroying enzyme (RDE). The samples were studied through an in-house enzyme-linked immunosorbent assay (ELISA) using whole influenza virus or recombinant proteins corresponding to nucleocapsid (N) protein and the receptor-binding domain of SARS-CoV-2 Spike (RBD) as antigens. We demonstrated that the heat inactivation of the naive serum samples of various hosts can lead to false-positive results, while RDE treatment abolished the effect of the non-specific binding of IgG antibodies to the viral antigens. Furthermore, RDE also significantly decreased the level of virus-specific IgG antibodies in SARS-CoV-2 and influenza-immune sera of humans and animals, although it is unknown whether it actually removes true virus-specific IgG antibodies or only non-specifically binding artifacts. Nevertheless, we suggest that the RDE treatment of human and animal sera may be useful in preventing false-positive results in various immunoassays, while also neutralizing infectious virus, since the standard protocol for the use of RDE also includes heating the sample at 56 °C.