Performance estimation of two in-house ELISA assays for COVID-19 surveillance through the combined detection of anti-SARS-CoV-2 IgA, IgM, and IgG immunoglobulin isotypes

Abstract

The main objective of this study was to estimate the performance, under local epidemiological conditions, of two in-house ELISA assays for the combined detection of anti-SARS-CoV-2 IgA, IgM, and IgG immunoglobulins. A total of 94 serum samples were used for the assessment, where 44 corresponded to sera collected before the pandemic (free of SARS-CoV-2 antibodies), and 50 sera were collected from confirmed COVID-19 patients admitted to the main public hospital in the city of Valdivia, southern Chile. The Nucleocapsid (Np) and the receptor-binding domain (RBD) proteins were separately used as antigens (Np and RBD ELISA, respectively) to assess their diagnostic performance. A receiver operating characteristic (ROC) analysis was performed to estimate the optical density (OD) cut-off that maximized the sensitivity (Se) and specificity (Sp) of the ELISA assays. Np ELISA had a mean Se of 94% (95% CI = 83.5–98.8%) and a mean Sp of 100% (95% CI = 92.0–100%), with an OD 450 nm positive cut-off value of 0.88. On the other hand, RBD ELISA presented a mean Se of 96% (95% CI = 86.3–99.5%) and a mean Sp of 90% (95% CI = 78.3–97.5%), with an OD 450 nm positive cut off value of 0.996. Non-significant differences were observed between the Se distributions of Np and RBD ELISAs, but the latter presented a significant lower Sp than Np ELISA. In parallel, collected sera were also analyzed using a commercial lateral flow chromatographic immunoassay (LFCI), to compare the performance of the in-house ELISA assays against a commercial test. The LFCI had a mean sensitivity of 94% (95% CI = 87.4–100%) and a mean specificity of 100% (95% CI = 100–100%). When compared to Np ELISA, non-significant differences were observed on the performance distributions. Conversely, RBD ELISA had a significant lower Sp than the LFCI. Although, Np ELISA presented a similar performance to the commercial test, this was 2.5 times cheaper than the LFCI assay (labor cost not considered). Thus, the in-house Np ELISA could be a suitable alternative tool, in resource limited environments, for the surveillance of SARS-CoV-2 infection, supporting further epidemiological studies.

The Anti-SARS-CoV-2 IgG1 and IgG3 Antibody Isotypes with Limited Neutralizing Capacity against Omicron Elicited in a Latin Population a Switch toward IgG4 after Multiple Doses with the mRNA Pfizer–BioNTech Vaccine

“…The levels of IgG4 remain at high levels for a long period and seem to not be affected either by a subsequent booster dose (e.g., bivalent vaccine) or by breakthrough infections. (5) Immunocompromised subjects, such as those with IBDs receiving treatment with immunomodulators, also induce IgG4 after the third dose, although these antibody levels have a limited effect on their immune system's neutralizing capacity. Further studies are needed to elucidate the mechanisms that underlie this class switch and determine its potential negative effect on people with comorbidities or immunosuppression, or who are at an older age.…”

“…Since the beginning of the COVID-19 pandemic, numerous quantitative/qualitative serological assays were developed across the globe as tools for detecting the presence of specific antibodies against SARS-CoV-2 [ 3 , 4 , 5 , 6 ]. As part of our contribution, our research group developed in-house serological assays to detect IgG antibodies in sera or plasma from individuals with COVID-19 in Puerto Rico [ 7 ].…”

Diseño factorial completo de experimentos para optimizar un ELISA que detecta anticuerpos anti-RBD del SARS-CoV-2

“…El ensayo inmunoabsorbente ligado a enzimas (ELISA) es un método analítico de uso común basado en la unión específica antígeno-anticuerpo. Este ensayo ha sido de importancia trascendental en la medicina humana, para detectar tanto antígenos como anticuerpos, y ha tenido gran aplicación en el diagnóstico in vitro debido a su alta especificidad y sensibilidad (Peng et al, 2022). El ELISA jugó un rol crucial en la reciente pandemia causada por el coronavirus SARS-CoV-2, tanto para el estudio de la respuesta inmune a la enfermedad COVID-19, como para la evaluación de la inmunogenicidad de las vacunas que se generaron para proteger a las personas de la enfermedad grave y la muerte (Ashmawy et al, 2022;Ramírez-Reveco et al, 2023).…”