Serological test to assess presence of immunoglobulin isotypes targeted against antigens routinely used in COVID-19 diagnosis

A fascinating new preprint discusses the potential for customized serologic testing in individuals who have or have not been vaccinated against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The results show the opportunity to maximize the cost-effectiveness of the test as well as its possibilities in understanding the immunogenicity and antigenicity of new strains of the virus and monitoring vaccine recipients.

Introduction

Several public health interventions, including mask-wearing, social distancing, hand hygiene, and even lockdowns, clubbed under the umbrella term “non-pharmaceutical interventions,” have been essential in the coronavirus disease 2019 (COVID-19) pandemic. In tandem, the rapid development and rollout of effective vaccines led to high vaccine coverage rates in most developed countries.

However, testing is important in identifying the spread of the virus and containing it. If the spread is not limited, more pathogenic variants may emerge, possessing vaccine-escape mutations. Thus, tests for SARS-CoV-2 antibodies must be developed that yield results rapidly while maintaining high sensitivity for multiple viral variants.

Serological tests have been found to vary in their performance between strains. As of now, they are not used to confirm the induction of immune antibodies with COVID-19 vaccines because they do not, in some cases, detect vaccine-elicited antibodies. Commercially available SARS-CoV-2 serologic tests are also expensive, making them unfit for extensive use.

What did the study show?

The current study, published on the Research Square* preprint server and under consideration at Scientific Reports, discusses a reliable enzyme-linked immunosorbent assay (ELISA) that can detect three isotypes of antibody against three SARS-CoV-2 antigens – the spike, the spike receptor-binding domain (RBDand the N-terminal domain (NTD) of the viral nucleocapsid (N) protein.

The spike and N antigens are mostly used for diagnosis as they are immunodominant antigens, but the RBD represents SARS-CoV-2 infection most accurately. They used each antigen at a concentration of 4 µg/ml, which was associated with the best distinction between pre-pandemic and pandemic profiles.

With the RBD at 0.6 µg/ml, either IgA or IgG antibodies showed equivalent responses, but not with IgA and IgM detection, which seemed to require the higher concentration.

Similarly, they determined the ideal dilution that allowed a clear demarcation of positive and negative samples between 1:50 and 1:100, for samples positive for the polymerase chain reaction (PCR). The background noise was minimal at these dilutions, and false positives were low.

The scientists tested three groups of individuals, those sampled before vaccination and found to have a positive test; those who had two doses of the vaccine but were seronegative before vaccination; and those who were first infected and then had two doses of the vaccine.

This showed higher antibody titers from vaccinated individuals with a history of prior infection compared to infection-naïve vaccinated individuals. The former were extremely variable, probably due to the difference in symptoms, the durability of immune responses depending on the collection time point, and inter-individual differences in responses to the vaccine and the infection.

Vaccination elicited greater increases in antibody titer when the initial titer was low, compared to when it was high.  

The secondary antibody dilution was similarly set at 1:10,000, and the incubation periods were set for each step to optimize the total time spent on the assay. The stability of the serum antibodies to temperature variations was tested using both inactivated and non-activated samples. This is necessary to ensure that the assay is reproducible since heat inactivation is recommended for all samples to decontaminate them.

They also examined the effect of pH variations on the antigen dilution buffer since this affects reproducibility. Measuring the reactivity of IgG to the Spike protein at different pH levels from 6.6 to 8.0. the optimal pH of 7.4 is recommended to ensure that positive samples are detected with high absorbance.

The cut-off values are a crucial element in diagnostic assays. Here again, they used two different techniques to determine the cut-off value in a range of 0.583 to 1.073 units of absorbance. The spike- and RBD-reactive IgGs showed very similar cut-offs, but not the N-NTD.

This is attributed to the large range of values for prepandemic IgG reactivity to N-NTD, caused by false positives, as well as the low sensitivity for this antigen because of the absence of antibodies against it in the PCR-positive samples of the assay.

IgA against the spike and RBD, but not the N-NTD; and IgM against the spike, but not the RBD, also showed high sensitivity and specificity.

Validation of the in-house assay showed 100% IgG reactivity to the spike, almost 80% and 50% to the RBD and N-NTD, respectively, in PCR-positive samples. IgG was detected in less than half the samples to all three antigens. Of non-diagnosed non-vaccinated individuals, less than one in seven were positive for IgG against any of the three antigens and less than 5% for all three.  

However, when symptomatic individuals from this group were separately examined, 17% were IgG-reactive to the spike and N-NTD, and 11% to the RBD. Similarly, 15% of asymptomatic individuals showed N-NTD reactivity, but less than 10% to the spike or RBD.

Interestingly, solitary anti-N-NTD reactivity was seen in seven individuals, which could be passed off as a cross-reaction or non-specific reactivity. But all of them seroconverted over the next five months, with anti-spike and anti-RBD reactivity and a 16% increase in anti-N-NTD IgG detection.

Immunologically, vaccinated and infected individuals showed differing reactivity patterns. Using either CoronaVac, an inactivated vaccine, or the adenovirus vector ChAdOx-1 nCoV19 vaccine, which presents all viral proteins vs. the spike protein, respectively, to the immune system, they found that both induced anti-spike IgG.

However, anti-RBD IgG was more strongly reactive in vaccinated individuals who tested positive for the virus by PCR. Neither vaccine induced a strong anti-N-NTD antibody response. Only ChAdOx-1 nCoV-19 induced a rise in anti-spike IgA in PCR-positive individuals and elicited an IgA response in seronegative individuals.

What are the implications?

This sensitive and specific assay can help detect the three immunoglobulin isotypes directed against the three antigens used to diagnose COVID-19. Such tests may help in serological surveillance, monitoring vaccination coverage, and determining how long protective immunity lasts against the virus following vaccination.

The current study focused on customizing the test for each stage, as required for its application, thus helping to reduce the consumption of reagents, samples, and the time required. In addition, its versatility allows it to be used for multiple different cohorts, making it highly efficient as well as permitting other mutant variants to be detected using the same assay. The cost is also significantly less.

The best performance from serologic assays is via multiple antigens, as in the present experiment. Here, not only is N-NTD strongly immunogenic, but both the spike and the RBD elicited highly specific responses. Anti-RBD antibodies are closely associated with a neutralizing response.

The use of all three isotypes helps improve the accuracy of diagnosis. However, the discrepancy between the high reactivity for IgM and IgG antibodies to the RBD and the high specificity for the RBD underlines the serious limitations of current knowledge.

Cross-reactive antibodies may come from earlier infection with the endemic seasonal human coronaviruses, with some homologous amino acids and conformational similarities. The dengue virus may also be partly responsible.

The serological test developed here allows the identification of diverse immune responses to various vaccine antigens, enabling a more precise understanding of the different immunization regimens across more specific populations.” Variants like the Omicron, which show antigen polymorphism, may escape detection by such tests. This could be due to the multiple mutations in the RBD coupled with reduced binding to human sera containing SARS-CoV-2 antibodies.

The use of a diagnostic test with a multiplex strategy using different antigens with different selective pressure has a clear advantage over tests using only RBD as antigen. Therefore, we believe that our multiplex ELISA test represents a valuable tool for defining more effective public health policies to fight this deadly contagious disease.”

*Important notice

Research Square publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.