Introduction
It is important to note that a major part of human immunity is reliant on antibody-antigen interactions. Antibodies are produced by a body to detect and eliminate antigens. The main antigens of SARS-CoV-2 are nucleocapsid proteins (N) and spike proteins (S). The study focuses on a more abundant antigen, which is membrane protein (M) since there is minimal research on M compared to N or S. SARS-CoV-2 virus has around 30kB RNA comprised of a single strand of a chain of nucleotides.
Background
In order to properly understand the selected study, it is critical to learn about the genetic material of SARS-CoV-2 virus. A single strand of DNA or RNA always has two ends: 5’ and 3’ terminals, which refer to a deoxyribose sugar’s carbon atom. At the 5’ end of the virus’s RNA, 2/3 of the genome is comprised of open reading frames or ORF. These are ORF1 and ORF2. They are the region, which can be translated. At the 3’ end, 4 key proteins are encoded. The order is spike (S), envelope (E), membrane (M), and nucleocapsid (N).
Methods
The methodological framework of the study is focused on two major approaches, which are ELISA and Western blot. ELISA is enzyme-linked immunosorbent assay, which, in layman’s terms, is done in three steps:
- Attach various antigens to a surface.
- Add antibodies to antigens for them to bind each other, and then wash away the unbound ones.
- Add a detector substrate for antibodies to see a signal.
Western Blot is a method of analyzing proteins.
- Proteins are separated by size
- Transfer of proteins
- Adding antibodies for detection
In the study, Western blotting was used for a assessing whether or not human antibodies, such as IgM or IgG, interact with SARS-CoV-2 virus’s M proteins. In Silico protein design method was used to recreate the recombinant structure of viral M protein. The protein was both purified and produced by the researchers.
ELISA was also used to see the interactional basis of human antibodies with the viral M protein. Lastly, the results were categorized in accordance with their specificity, sensitivity, negative predictive value, and positive predictive value.
Results
The results come in three parts, and firstly, the production and purification of recombinant M protein revealed that it has five parts, such as N-terminal (1-19), transmembrane region I or TMI (20-39), TMII (51-73), TMIII (78-100), and C-end (101-222). TM regions were removed and replaced with GGG since they are not relevant. The overall molecular mass of M protein is around 16500 Da, and consists of 149 amino acids.
Secondly, immunoreactivity of the recombinant SARS-CoV-2 M protein in Western Blot showed that IgM and IgG have a high degree of reactivity with SARS-CoV-2 M protein or M. Non-COVID-19 subject sera sample did not react with M thorough their IgM and IgG. A single band detection shows that recombinant M or purified M protein is pure and specific. IgM and IgG-based non-specific binding was not detected, which eliminates a potential of error.
Thirdly, immunoreactivity of the SARS-CoV-2 M protein in ELISA showed that IgM detected COVID19 sera with an specificity of more than 93%. Similarly, IgG detected COVID19 sera with an specificity of more than 96%. Both IgM and IgG were high specific and sensitive to M protein. Negative predictive value was 91.1% and 97.4% for IgM and IgG, respectively. Positive predictive value was 84.8% and 90.6% for IgM and IgG, respectively.
Discussion
It is important to note that the vast majority of tests are based on N and S protein detection. However, these two comprised only a small fraction of antigen elements delivered by SARS-CoV-2 virus. The most abundant antigen is M protein of the viral membrane, which is why including it is the tests can increase accuracy and reach of testing measures. Both IgG and IgM were responsive to M protein, which evidences its plausibility. All three proteins, M, N, and S, are codominant, which means they are fully recognizable.
The majority of current research efforts and studies are focused on the immune response mechanisms against S protein of the coronavirus. There is limited data on immune response on M protein despite its predominant abundance compared to other antigen elements. The given research provides a basis for future assessments on M protein’s role in testing and vaccination measures. M protein can be even considered as a better alternative than S protein for vaccine production due to its commonality among all coronavirus strains.
Conclusion
In conclusion, there are three types of proteins produced by SARS-CoV-2 virus, such as M protein, S protein, and N protein. Both ELISA and Western blotting methods of analysis show high specificity of IgG and IgM to M protein. M protein more abundant source of antigen and conserved across all coronaviruses. More efforts should be put on M protein studies.