Mass Antibody Detection: Can It be An Avenger for Infectivity War against COVID-19

The ongoing pandemic of COVID-19 has not only commenced a global health emergency but agitated various aspects of humanity. During this period of crisis researchers over the world have ramped their efforts to constrain the disease in all possible ways whether it is vaccination, therapy, or diagnosis. Since the spread of the disease has not yet elapsed sharing the ongoing research findings could be the key to disease control and management. An early and efficient diagnosis could leverage the outcome until a successful vaccine is developed. Molecular tests both in-house and commercial kits are preferably being used worldwide in the COVID-19 diagnosis. However, the limitation of high prices and lengthy procedures impede their use for mass testing. Keeping the constant rise of infection in mind search for an alternative test that should be cost-effective, simple, and suitable for large scale testing and surveillance is a need of an hour. One such alternative could be the immunological tests. Therefore, in the last few months deluge of immunological rapid tests has been developed and validated across the globe. The objective of the present review is to share the diagnostic performance of various immunological assays reported so far in SARS-CoV-2 case detection. The article consolidated the studies (published and preprints) related to the serological tests such as chemiluminescence, enzyme-linked and lateral flow-based point-of-care tests in COVID-19 diagnosis and updated the current scenario. This review will hopefully be an add-on in COVID-19 research and will contribute to congregate the evidence for decision-making.


What we know about this real-world devil
The world today has been amalgamated like never before to fight a common enemy to mankind, the pandemic disease namely Coronavirus. Coronaviruses (CoV)  Clinical manifestations of COVID-19 include fever, dry cough, fatigue, and pneumonialike features. The coronavirus is reported to interact with host epithelial cell receptor, angiotensin converting enzyme 2 (ACE2), found in outer surfaces of most of the human body organs leads to endocytosis into the host cell 3 . Severity of the disease occurs when COVID-19 virus (SARS-CoV-2) enters into the lungs through the oral or nasal passage, divides within the lung cells which lead to its death. Dead cells may generate an inflammatory response through IL-6, IL-1, and TNF-α etc., resulting in the consolidation of fluid in the alveolar and interstitial spaces, known as acute respiratory 4 distress syndrome (ARDS). The symptomatic clinical condition without ventilation may lead to death due to the insufficiency of oxygen in the vital organs thus multiple organ dysfunctions (kidney injury and cardiac arrest). Chances of this affecting the older age group are more due to their already present comorbidities.

Brief introduction of the current diagnosis
In the present scenario, while the vaccine is not available for COVID-19 and there is no short and sure treatment, rapid and extensive diagnosis could be the way to reach the grass-root level of the disease and break the widespread chain of this deadly COVID-19 infection 4 . It will have a relevant role in the containment of the disease until a suitable therapy or vaccine strategy is attained. The conventional diagnosis is based on the clinical history and the radiographic findings of the chest but due to the lack of trained individuals in virus imaging and invasiveness of the process, it is routinely practiced only in the initial phase of the epidemic in China. In several areas temporary CT scans have been utilized for clinical diagnosis for false-negative results. These are non-invasive tests, however, was low on specificity, as lung images overlap with any common viral disease. A recent study reported a confirmed case of COVID-19 with no signs of pneumonia in chest CT image 3 . Moreover, CT scans are expensive and constrained in the research labs and big hospitals requiring technical know-how.
From a worldwide perspective, the most popular and gold standard technique which is being used for COVID-19 diagnosis globally is an initial symptomatic analysis followed by molecular-based nucleic acid detection from sputum, nasal and mouth swab and 5 serum . The COVID-19 bears a single-stranded RNA genome of approximately 30,000 nucleotides. The nucleic acid testing of COVID-19 is based on reverse transcription polymerase chain reaction (RT-PCR) technique 5 . Several conserved COVID-19 regions have been targeted in the molecular tests such as RNA dependent RNA polymerase gene (RdRP gene), nucleocapsid protein gene (N gene), and envelop protein gene (E gene). Since this test can amplify even low levels of the virus and detect the disease at an early stage, this has been the most used and reliable detection method. However, the performance of RT-PCR is largely depends on the target viral RNA selected and the primers used. Therefore, the World Health Organization (WHO) time to time published the standard protocol for the parity of the assay. Secondly, variations in the viral load are noticed in different stages of disease manifestations and the biological samples used 6 . Despite a large number of RT-PCR based tests is being conducted throughout the globe its accessibility to reach centers of testing from remote areas restricts its mass appeal. Moreover, they are expensive, equipment-based, time taking, and require developed molecular biology expertise. However, the results are preliminary and most of the in-house PCR tested worldwide have a high false-negative rate in COVID-19 diagnosis 7 . Missed diagnosis of a large number of clinically suspected individuals may promote the spread of the virus that may ultimately lead to faster disease progression.
Moreover, in many cases molecular testing often requires more than one test for disease confirmation 8 . RT-PCR is valuable in the initial phase of infection when the virus is present in the body, however, it could not identify the past, recovered, and asymptomatic infections. Most of the countries are under lockdown for almost two months now, it is essential to have a vigorous door to door testing to counter the 6 pandemic. Molecular biology-based tests in such cases will become highly cumbersome and should be eased with easier and quicker tests.
Rapid diagnosis to test as much as we can at this point in time may be considered the only hope to fight the disease. As emphasized by the chief of WHO on 16 th March just three days after declaring COVID-19 a pandemic to "test, test and test". The Foundation for Innovative New Diagnostics (FIND), Geneva, Switzerland, has published the first result of independent evaluation and clinical performance of five molecular test kits on 16 th April. 100% sensitivity was obtained by all the kits with 50 COVID-19 positive samples and 97-100% specificity achieved with 100 negative samples 9 . However, there may be asymptomatic or infected patients, who may have cleared the viral load without detection, thus an RT-PCR will fail to test such cases. Therefore, it is important to develop and introduce a rapid and point-of-care test to detect COVID-19 cases and carriers. The antibody-based tests can play an important role in the restraint of COVID-19, facilitate the rapid execution of a control strategy that limits the spread of the disease. The National Health Commission of China in the seventh edition of its novel coronavirus pneumonia diagnosis and treatment plan recently recommended the use of IgM/IgG antibodies testing for suspicious cases 10 . Additionally, the convalescent patients will have to be monitored closely for a long-term period, which will involve repeated testing for the presence of antibodies against the disease. For the presence of antibodies, accuracy will be needed, as they will serve as tests of cure. Since most of the countries have permitted the use of antigen or antibody-based serological tests the sensitivity and specificity of these tests against COVID-19 are being reported throughout the world. With the dynamics of the disease changing at the speed of its 7 spread, it seems highly essential to get all the information at a glance related to the performance of serological tests reported worldwide to pace our health care needs. This review aims to have a compilation of the performance of various serological assays across the globe for the detection of COVID-19 pandemic.

Key determinants of immunoassay
Although molecular tests are the gold standard and very specific in early COVID-19 detection its reach to large scale diagnosis is constrained in terms of its cost, feasibility, and rapidness. Immunological assays could be a good complement in this regard.  11 . The trimeric S protein (~180 kDa monomer) is postulated to be the first viral fraction to bind with host cell receptors through the receptor-binding domain (RBD) of S1 subunit and helps in the 8 fusion of virus and host membranes through S2 subunit. M protein plays its role in viral morphogenesis and budding through its glycosylated N terminal domain, three transmembrane domains, and a long C terminal domain. E protein is the smallest structural protein that constitutes the viral assembly, release, and pathogenesis. The N protein is the most abundant viral protein (~40 kDa) thus could be an ideal candidate for diagnosis 12 . A high immune response has been demonstrated against this protein and detected in blood and urine samples both. Despite its abundance, S proteins have also been used in COVID-19 diagnosis because of its specificity 13  residue. Interestingly, no antibodies were detected to E protein 15 . In another proteome microarray, 29 convalescent sera of COVID-19 were used to demonstrate the IgM/IgG response against 18 SARS-CoV-2 protein constructs. The result showed 100% antibody response mainly for N and S1 protein where S1 proved to be the best in differentiating COVID-19 patients from controls 16  Additionally in critical COVID-19 patients the results suggested a significantly higher concentration of IgG than mild and moderate cases 19 . A serological study in Italy observed 100% sensitivity for IgG than 88% for IgM at day 12 20 . A study from China reported 97.7% and 95.6% sensitivity and 95.2% and 96.6% specificity of IgM and IgG antibodies respectively 21 . 100% sensitivity and specificity were achieved in USA with 10 commercial IgG CLIA test at day 17 from the disease onset 22 . In another study, seropositivity in confirmed COVID-19 patients was found within 7-12 days of disease onset and continues with disease progression. The overall specificity of IgM and IgG with non-COVID-19 suspected cases, other diseases, medical staff, and healthy controls were found to be 97% 8

Antibody ELISA in COVID-19 diagnosis
Since conventional ELISA is a cumbersome and time-consuming process diagnostic companies provide precoated ELISA either with EUO or IVD labeling to detect virusspecific antibodies in human samples. However, the sensitivity and specificity of the ELISA kit largely relies on the type of viral protein used. Nevertheless, the seroconversion of antibodies in COVID-19 patients depends on the onset of symptoms, therefore, the day on which the test is being conducted should be an important consideration. Liu 33 . In contrast a cohort study in Hong Kong suggested the early seroconversion of IgG than IgM antibodies in confirmed cases. Moreover, antibodies after 14 days of disease onset showed 100% sensitivity for IgG and 94% for IgM against RBD antigen whose seropositivity was observed earlier than N protein 34 Table 3. Figure 1 depicts the diagnostic performance of immune assays throughout the countries.

A hunt for asymptomatic cases
The world has faced bigger pandemics in the past. The virus has infected humans before in the form of SARS and MERS. Then why the scare and why call the pandemic unprecedented? The severity of a disease depends on its transmission rate and death rate. Unlike SARS (R0=), the transmission rate of COVID-19 is suspected to be very high especially through asymptomatic cases, with an average R0 value of around 3.28 54 .
Thereby increasing the chances of spread due to these undetected asymptomatic cases

Conclusion
Like any previous pandemic, the answer to curing the disease will be an appropriate vaccine strategy. However, the solution to managing COVID-19 till then cannot be limited to any one thing, it has to be a holistic approach, like the WHO Director-General