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Can Multiplex ELISA Replace or Reduce Burden of PRNT? A View 
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The COVID-19 pandemic has led to a dramatic loss of human life worldwide. The economic and social disruption caused by the pandemic is devastating with an unprecedented challenge to public health, food systems, and the world of work. Causing nearly 169 million infections and over 3.5 million deaths until late May 2021, the virus that causes this infectious disease is Severe acute respiratory syndrome (SARS) i.e. SARS-CoV-2.

Nevertheless, in many countries, testing is still minimal or rationed, as the testing capacity is overwhelmed by the extent of the outbreak. In such a case, neither mild cases nor asymptomatic cases are being tested by RT-qPCR unless they are in direct contact with known cases.

Immune responses to SARS-CoV2 in the early weeks of the infection have been reported worldwide using various techniques such as ELISA, PRNT, etc. Even in people tested with SARS-CoV-2 infection, many might have a negative result at the time of testing due to low viral load. Therefore, serological tests such as the ELISA and the Luminex Microsphere Assay have been positively associated with antibody concentration and can be an important complementary approach. Because ELISA tests are able to test antigens and antibodies, they are considered flexible tools by medical professionals, and laboratories from all over the world.

These rapid testing kits can not only be used to differentiate between the ones infected vs vaccinated but also, to generate data-driven decision-making with sufficient knowledge. A powerful antibody test also has the potential to be used as a form of Covid passport, which can enable people to travel or return to work, providing personal protection and information for the same. Similarly, measuring Antibody levels, either in the form of qualitative or quantitative (S, M, N, E) will be the future. Given this, India can be a front runner in this area. The ability to provide quicker results can help track the spread of disease across the nation, and in turn protect citizens.

However, serological tests with one antigen have been found to generate false-negative data in about 8-10% of the populations tested, thus a need for alternative methods for screening in the context of epidemiological surveys.

The antibody response generated after SARS-CoV-2 infection consists of multiple isotypes such as IgG, IgM, IgA. These isotypes target several proteins on the virus, including the spike protein, RBD, and nucleoprotein.

Premas Biotech has optimized the parameters for the expression and purification of spike S1, M, E, and N proteins. Here, we developed an assay using the ELISA to simultaneously detect IgG antibodies to the highly pathogenic human coronaviruses SARS-CoV2 using the four viral antigens for the SARS-CoV-2. The median duration between COVID-19 symptoms onset and sample collection is between <6 to >22 days.

Multiplexing or combining two antigens to deliver a result that can have high benefits as seen by a small 80 convalescent RT-PCR positive patient data generated. This interesting data on a relatively small sample of patients allows us the opportunity to build multiplexed ELISA tests for serological surveillance studies and would be able to bring down the false-negative data in a greater population.

ELISA data shows the binding to the Spike and M proteins of SARS CoV-2 to the patients’ sera (Figure 1). We used the (Mean+2xSD) formula to calculate the cut-off by calculating the mean of OD of the COVID-19 negative samples for each of the recombinant proteins tested. We added to the value obtained two times the standard deviation. The result obtained was considered as the cut-off for each antigen. Mean OD in the COVID-19 group were 1.4 ± 0.29 and 0.94 ± 0.23, for Spike and M proteins respectively. These values were 0.63 ± 0.07 and 0.39 ± 0.05 for the Spike and M proteins in the negative control group, respectively. Combining S and M protein data for the patients, we observed that there were patients who have had a lower response to S1(ODcut off; 0.52). Our data showed that about 14% of the patients in this serological study would have been false negative or missed the positive results if they had been tested just for S protein.

Figure 1

Next, to determine IgG levels for S and M antigens, we selected 12 samples from the early phase of COVID-19 symptoms (≤14 days) and 12 others from later stages (>14 days). Results showed that the IgG response of samples from the earlier phase was higher than those from the later phase but was not significant. At early phases (≤14 Days), 11/12 and 10/12 of the tested samples presented a two-fold increase in cut-off values in IgG levels against Spike and membrane proteins, respectively. For the later phases, the values were lower in >50% cases. These results suggest loss of IgG in the later phase. This observation stands for both the Spike and the membrane proteins (Table 1).

Table 1

To measure IgG levels by using all four antigens simultaneously, we have randomly selected 4 samples. The results of multiplex ELISA by using all four antigens (SEMN) showed a high IgG response in patients' samples (N=3) compared with S, M, and N antigens of SARS-CoV-2 (Figure 2).

Figure 2

By Reeshu Gupta
Lead-Content Generation

05 May 2021