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A qualitative IgG ELISA for detection of SARS-CoV-2-specific antibodies in Syrian hamster serum samples.

ABSTRACT: This protocol describes an indirect enzyme-linked immunosorbent assay for qualitative detection of IgG antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Syrian hamster serum samples. We describe the preparation of inactivated virus antigens and the negative control antigen and the use of antigen-coated microtiter plates to detect SARS-CoV-2-specific antibodies from SARS-CoV-2-infected hamsters, including the criteria for differentiating positive versus negative reaction. The limited batch-to-batch variability of this assay has been verified with two batches of independently prepared antigens. For complete details on the use and execution of this protocol, please refer to Mohandas et al. (2021).

SUBMITTER: Shete A

PROVIDER: S-EPMC8108381 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

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A qualitative IgG ELISA for detection of SARS-CoV-2-specific antibodies in Syrian hamster serum samples.

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