Comparative analyses of SARS-CoV-2 binding (IgG, IgM, IgA) and neutralizing antibodies from human serum samples

Abstract

A newly identified coronavirus, named SARS-CoV-2, emerged in December 2019 in Hubei Province, China, and quickly spread throughout the world; so far, it has caused more than 49.7 million cases of disease and 1,2 million deaths. The diagnosis of SARS-CoV-2 infection is currently based on the detection of viral RNA in nasopharyngeal swabs by means of molecular-based assays, such as real-time RT-PCR. Furthermore, serological assays detecting different classes of antibodies constitute an excellent surveillance strategy for gathering information on the humoral immune response to infection and the spread of the virus through the population. In addition, it can contribute to evaluate the immunogenicity of novel future vaccines and medicines for the treatment and prevention of COVID-19 disease. The aim of this study was to determine SARS-CoV-2-specific antibodies in human serum samples by means of different commercial and in-house ELISA kits, in order to evaluate and compare their results first with one another and then with those yielded by functional assays using wild-type virus. It is important to identify the level of SARS-CoV-2-specific IgM, IgG and IgA antibodies in order to predict human population immunity, possible cross-reactivity with other coronaviruses and to identify potentially infectious subjects. In addition, in a small sub-group of samples, a subtyping IgG ELISA has been performed. Our findings showed a notable statistical correlation between the neutralization titers and the IgG, IgM and IgA ELISA responses against the receptor-binding domain of the spike protein. Thus confirming that antibodies against this portion of the virus spike protein are highly neutralizing and that the ELISA Receptor-Binding Domain-based assay can be used as a valid surrogate for the neutralization assay in laboratories that do not have biosecurity level-3 facilities.

Keywords: ELISA; Human samples; Micro-neutralization; Receptor-binding domain; SARS-CoV-2.

Fig. 1

The correlation plot associated to the measured coefficients of Spearman's rank correlation. The magnitude of the coefficient is represented by circles and a color gradient: the larger the area of the circle and the more intense the tone of the color, the greater the correlation. The direction of the correlation is indicated by the color scale: blue tones for positive correlations and red tones for negative correlations. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

A) Analysis of the ROC curve referred to the test set proved that the results of the EN model attained high accuracy in predicting the MNT values. Measurement of the area under the curve, AUC = 90.7%, supported this conclusion; B) Summary table of ROC analysis; C) Error matrix.

Fig. 3

Percentage of detection of IgG1, IgG2, IgG3 and IgG4 in all 14 human samples positive on MN assay. Each column represents the contribution, in terms of percentage, each IgG subclasses versus SARS- CoV- 2 RBD. Error bars showing the variance of sample proportion.

Fig. 4

Log transformed MNTs before and after the treatment with the goat anti-human IgA antibodies; t-Test shows a significant decrease in the MN titers for those samples with high neutralizing titers.