*3.3.1 Automated CLIA*

The magnetic particles-based CLIAs are the gold standard in clinical diagnostics for the automated high-throughput analysis of many disease biomarkers on a random-access CLIA analyzer. The desired throughput can be customized from low- to high-throughput by selecting an appropriate CLIA analyzer. Many IVD companies have developed several SARS-CoV-2 serology tests for the detection of IgG and IgM. Most of these assays have an overall assay duration of less than 30 min and employ SP, NP, or both for the detection of anti-SARS-CoV-2 Ab.

The DiaSorin LIAISON® SARS-CoV-2 S1/S2 IgG assay [34, 35], used on the LIAISON® XL Analyzer, detects the anti-SARS-CoV-2 IgG qualitatively in human serum or plasma (sodium heparin, lithium heparin, and potassium EDTA). The assay employs the specific recombinant S1 and S2 antigens coated magnetic particles, which detect the anti-SARS-CoV-2 IgG in the patient sample. This is followed by subsequent binding to mouse monoclonal Ab to human IgG linked to an isoluminol derivative (isoluminol-Ab); adding the starter reagents to induce flash chemiluminescence (CL) reaction; and, measuring the CL signal of isoluminol-Ab conjugate via a photomultiplier. The assay employs two calibrators, one containing low and another having high anti-SARS-CoV-2 IgG levels. The assay has a PPA of 97.6% in samples taken from COVID-19 subjects ≥15 days after diagnosis by RT-PCR. DiaSorin also developed LIAISON® SARS-CoV-2 S1/S2 IgM assay for the qualitative detection of anti-SARS-CoV-2 IgM in human serum or plasma (sodium heparin, lithium heparin, and dipotassium EDTA). The assay principle is similar to that of IgG assay except that magnetic particles were coated with spike receptorbinding domain (RBD) antigen and mouse monoclonal IgG antibodies were linked to an isoluminol derivative, N-(4-Amino-Butyl)-N-Ethyl-Isoluminol (ABEI isoluminol-Ab conjugate). It employs two calibrators, one containing anti-SARS-CoV-2 human IgM monoclonal Ab while the other has no IgM. The PPA to PCR was 92.6% in samples taken from COVID-19 subjects between 15 and 30 days after PCR diagnosis.

The Elecsys anti-SARS-CoV-2 S cobas® [34, 36] is an electrochemiluminescent IA developed by Roche for the qualitative and semi-quantitative detection of Ab against the SP RBD in human serum and plasma (lithium heparin, dipotassium-EDTA (K2-EDTA), tripotassium EDTA (K3-EDTA), and sodium

#### In Vitro *Diagnostics for COVID-19: State-of-the-Art, Future Directions and Role in Pandemic… DOI: http://dx.doi.org/10.5772/intechopen.97775*

citrate). The IA is performed on cobas® e analyzers and has a total assay duration of just 18 min. It employs double-antigen sandwich principle, where the antigens present in the reagent capture mainly anti-SARS-CoV-2 IgG but also IgM and IgA. The assay procedure comprises of two incubations and a measurement step. The 1st incubation of the sample with biotinylated SARS-CoV-2 SP RBDspecific recombinant antigen and biotinylated SARS-CoV-2 SP RBD-specific recombinant antigen labeled with a ruthenium complex leads to the formation of a sandwich complex. It is followed by the 2nd incubation, during which the addition of streptavidin-coated magnetic particles binds to the sandwich complex via streptavidin-biotin interaction. Subsequently, the reaction mixture is aspirated into the measuring cell where the electrode magnetically captures the magnetic particles and the unbound substances are removed. Finally, a voltage is applied to the electrode, which induces the CL signal that is measured by a photomultiplier. The results are determined using a calibration curve with 2-point calibration and a master curve. The assay demonstrated a PPA to PCR of 96.6% in samples taken from COVID-19 subjects ≥15 days after PCR positive result and a NPA to PCR of 99.98%.

Another prospective assay is the IDS SARS-CoV-2 IgG assay developed by Immunodiagnostic Systems, United Kingdom (UK), which detects the anti-SARS-CoV-2 IgG antibodies qualitatively in human serum and plasma (lithium heparin, K3-EDTA, and sodium citrate). The assay involves the incubation of magnetic particles coated with recombinant SARS-CoV-2 NP and SP antigens with the patient sample (4 μL), which is followed by a wash step and subsequent incubation with anti-SARS-CoV-2 Ab labeled with acridinium. The magnetic particles, having the specific immune complexes, are then captured by a magnet, and a wash step removes the unbound substances. Subsequently, the addition of trigger reagents induces the CL signal that is measured by a photomultiplier. The IA employs two calibrators, one calibrator having low anti-SARS-CoV-2 IgG level while other calibrator has high anti-SARS-CoV-2 IgG level. The assay demonstrated a PPA to PCR of 97.6% in samples taken from COVID-19 patients ≥15 days after symptoms onset and a PPA to PCR of 100% in samples taken from COVID-19 subjects ≥15 days after PCR method. The NPA to PCR was 99.6%.

Of interest is the SNIBE's MAGLUMI 2019-nCoV IgM/IgG assay [37], which employs the 2019-nCoV recombinant antigen, expressing the full-length SP and NP. The assay employs two separate cassettes; one cassette detects IgM via a capture CLIA while another cassette detects IgG via an indirect CLIA. The CL signal detection mechanism is similar to that of DiaSorin. It employs two calibrators and two controls both for the IgG as well as IgM assays. One calibrator and control have high anti-SARS-CoV-2 IgG/IgM levels, while another calibrator and control have low anti-SARS-CoV-2 IgG/IgM levels. The results are generated by a calibration curve generated by 2-point calibration for a specific SNIBE's analyzer and a master curve. The IgG and IgM assays demonstrated PPAs to PCR of 100% and 77.46%, respectively, in samples taken from COVID-19 patients ≥15 days post symptoms onset. The combined PPA to PCR was 93.94% in samples taken from COVID-19 patients ≥15 days post symptoms onset.

There are several other CLIAs developed by Siemens, Abbott, Beckman Coulter, Ortho Clinical Diagnostics, etc., which employ similar assay formats and have high analytical performance.

### *3.3.2 ELISA*

Many IVD manufacturers have developed several CE-certified ELISA kits for the detection of anti-SARS-CoV-2 IgG and IgM antibodies. The most prominent ELISA

kits from Euroimmun, InBios, Wantai, Epitope Diagnostics, and Thermo Fisher Scientific, have been approved by FDA under EUA.

The most widely used ELISA kit is the EUROIMMUN Anti-SARS-CoV-2 ELISA (IgG) kit [35, 38], which enables the qualitative detection of anti-SARS-CoV-2 IgG antibodies in human serum and plasma (K+ -EDTA, Li+ -heparin, and Na<sup>+</sup> -citrate). The test kit contains microplate strips, where each strip has 8 break-off reagent wells that are pre-coated with S1 domain of SP of SARS-CoV-2, which is expressed recombinantly in the human cell line HEK 293. The assay procedure involves incubating reagent wells with diluted patient sample (diluted 1:101 in sample buffer), which leads to the binding of anti-SARS-CoV-2 IgG antibodies (and also IgA and IgM) with the coated viral Ag. It is followed by subsequent incubation with HRP labeled anti-human IgG detection Ab and TMB substrate reaction. The absorbance of the colored solution after stopping the enzyme-substrate reaction with a stop solution is measured at the wavelength of 450 nm with a reference wavelength of 620–650 nm. The provided two controls, i.e., a positive and a negative control, and calibrator must be used with each run. The overall assay duration is about 2 h and 15 min. The test is evaluated by calculating a ratio of the OD of the control/patient sample over the OD of the calibrator. The results are interpreted as negative, borderline, or positive if the ratio is <0.8, 0.8–1.1, or > 1.1, respectively. It is recommended to retest the patient after 1–2 weeks if he has a borderline result. The assay demonstrated a PPA to PCR of 100% in samples taken from COVID-19 patients ≥21 days post onset of symptoms and a PPA to PCR of 81.1% in samples taken from COVID-19 patients ≥11 days post PCR confirmation. The NPA to PCR was 98.6%. The independent validation study by Frederick National Laboratory for Cancer Research (FNLCR) determined the PPA to comparator method of 90% and NPA of 100%.

The SCoV-2 Detect™ IgG ELISA from InBios International, Inc., USA employs indirect ELISA for the qualitative detection of anti-SARS-CoV-2 IgG antibodies in diluted serum samples (diluted 1:100 in sample dilution buffer). The assay procedure and steps are similar to that of Euroimmun ELISA kit, and the overall assay duration is about 2 h. The kit has a positive and a negative IgG control and a cut-off IgG control. All the controls should be run whenever an assay is performed. The assay achieved a PPA to PCR of 100% in samples taken from COVID-19 subjects ≥22 days post onset of symptoms and a PPA to PCR of 100% in samples taken from COVID-19 subjects 0–28 days post PCR confirmation. The PPA to PCR was 95.45% in samples collected from suspects ≥15 days post onset of symptoms. The NPA to PCR was demonstrated to be 98.95%. The independent validation study by FNLCR showed both PPA and NPA to the comparator method to be 100%.

InBios has also developed the SCoV-2 Detect™ IgM ELISA that enables the qualitative detection of anti-SARS-CoV-2 IgM antibodies in human serum and plasma (K2-EDTA) from individuals that are 7–64 days post symptoms onset. The assay procedure is similar to that of Euroimmun and InBios IgG kits, except that detection is done by HRP labeled anti-human IgM. The IA involves dilution of the patient sample, i.e., 1:100 in sample dilution buffer, and has an assay duration of about 2 h. It employs a positive and a negative IgM control, and a cut-off IgM control, which must be run for every assay. The assay achieved a PPA to PCR of 93.75% in samples taken from COVID-19 subjects ≥15 days post onset of symptoms. The NPA to PCR was demonstrated to be 98.95%. The independent validation study by FNLCR showed PPA and NPA to comparator method of 96.7% and 98.8%, respectively.

Bio-Rad has developed the Platelia SARS-CoV-2 Total Ab ELISA that enables the qualitative detection of total Ab (IgM/IgG/IgA) against SARS-CoV-2 in human serum and plasma (K2-EDTA, K3-EDTA, lithium heparin, acid citrate dextrose,

#### In Vitro *Diagnostics for COVID-19: State-of-the-Art, Future Directions and Role in Pandemic… DOI: http://dx.doi.org/10.5772/intechopen.97775*

or sodium citrate) [39, 40]. It is a one-step antigen capture format that employs the addition of pre-diluted patient samples to recombinant SARS NP coated wells, and the simultaneous addition of HRP labeled recombinant SARS NP. The mixture is incubated for 1 h at 37°C that leads to the formation of immune complex. Thereafter, the TMB substrate is added, and after 30 min, the colorimetric reaction is stopped by adding a stop solution. The optical density is read by a spectrophotometer at 450 nm with a reference wavelength of 620 nm. The assay employs a positive control, a negative control, and a cut-off control that should be run for each assay. The positive and cut-off controls comprise rabbit polyclonal anti-SARS NP antibodies in human serum and buffer, respectively, with other components. The analysis of results is similar to that of Euroimmun IgG ELISA. The assay has a PPA to PCR of 100% in samples taken from COVID-19 suspects ≥15 days post onset of symptoms while the NPA to PCR was 98.3%. A similar ELISA kit is the WANTAI SARS-CoV-2 Ab ELISA that enables the qualitative detection of total antibodies against SARS-CoV-2 in human serum and acid citrate dextrose plasma. The PPA to PCR was 98.72% in samples taken from COVID-19 suspects ≥15 days post onset of symptoms while the NPA to PCR was 98.3%. The FNLCR's independent validation showed PPA and NPA to comparator method of 96.7% and 97.5%, respectively.

A novel ELISA is the cPassTM SARS-CoV-2 Neutralization Activity Detection Kit from GenScript USA, Inc., which detects qualitatively the total anti-SARS-CoV-2 neutralization Ab in human serum and K2-EDTA plasma (diluted 1:9 in sample dilution buffer) in just less than 1 h 15 min. The test mimics the virus-host interaction in a test tube or microplate by coating the surface of the well with human ACE2 and analyzing its specific binding to the purified recombinant SARS-CoV-2 SP RBD protein conjugated to HRP (HRP-RBD). The specific interaction between human ACE2 and HRP-RBD is blocked when the neutralization Ab against SARS-CoV-2 SP-RBD are present in the patient sample. The patient samples and controls are diluted and incubated with HRP-RBD so that the neutralization Ab could bind to HRP-RBD. It is followed by the addition of mixture to human ACE2 protein coated capture plate, where the unbound HRP-RBD and HRP-RBD bound to non-neutralizing Ab is captured on the plate. The neutralization Ab HRP-RBD immune complexes in the supernatant are removed during the washings. Subsequently, the TMB substrate is added, and the enzymatic reaction is stopped by adding a stop solution. The absorbance of the solution is read at 450 nm in a microplate reader. The assay employs a positive and a negative control that should be run for each assay. It has PPA and NPA of 100% with the comparator Plaque Reduction Neutralization Test at 50% viral neutralization (PRNT50) and 90% viral neutralization (PRNT90). The PRNTs employ the SARS-CoV-2 virus (WA01/2020 isolate).

## *3.3.3 Rapid LFIA*

Numerous rapid LFIA-based POC tests have been developed to detect and differentiate anti-SARS-CoV-2 IgM and IgG antibodies produced in individuals after SARS-COV-2 infection. They provide the test results in less than 20 min using only a few microliters of the sample.

The COVID-19 IgG/IgM Rapid Test Cassette (Whole Blood/Serum/Plasma) test from Healgen Scientific LLC, USA, is rapid LFIA for the qualitative detection and differentiation of anti-SARS-CoV-2 IgM and IgG antibodies in human venous whole blood, serum, and plasma from anticoagulated blood (Li<sup>+</sup> heparin, K2-EDTA, and sodium citrate) [41]. It employs anti-human IgG, anti-human IgM and rabbit IgG coated on a nitrocellulose strip at the test line IgG, test line IgM and control line C,

respectively. The colloidal gold particles conjugated to recombinant SARS-CoV-2 S1 antigen (COVID-19 conjugates) are stored in the burgundy-colored conjugate pad. The assay procedure involves the addition of patient sample and assay buffer to the sample well. The presence of anti-SARS-CoV-2 IgM and/or IgG antibodies in the sample will lead to the formation of immune complex with COVID-19 conjugates, which will migrate through nitrocellulose membrane via capillary action and forms a burgundy-colored band at the test line IgM and/or IgG. The absence of colored test bands indicates a negative result. The control line C serves as a procedure control, which will change from blue to red if the proper volume of sample has been added and membrane wicking has occurred. The manufacturer recommends using positive and negative controls as a good laboratory practice to confirm the test result. These are not provided by the company but need to be identified by the laboratory themselves. The assay takes only 10 min and must be read visually within 15 min. The PPA and NPA for the detection of anti-SARS-CoV-2 IgG and IgM antibodies were 96.7% and 98%; and, 86.7% and 99%, respectively. The independent clinical study determined the overall PPA of 100% and NPA of 97.5%.

Another rapid test, the BIOTIME SARS-CoV-2 IgG/IgM Rapid Qualitative Test from Xiamen Biotime Biotechnology Co., Ltd., China, detects and differentiates anti-SARS-CoV-2 IgM and IgG antibodies in human serum, potassium EDTA venous whole blood and plasma (potassium EDTA) [35]. It takes only 20 min and must be read visually within 30 min. The company has SARS-CoV-2 IgG/IgM Control Set, which can be purchased separately by customers. The PPAs to PCR for the detection of anti-SARS-CoV-2 IgG and IgM antibodies in serum and plasma were 100% in samples collected from COVID-19 patients ≥15 days post onset of symptoms. The NPA to PCR for the detection of anti-SARS-CoV-2 IgG and IgM antibodies in serum and plasma were 98.46% and 100%, respectively. The FNLCR validation study demonstrated sensitivity and specificity for anti-SARS-CoV-2 IgM and IgG to be 100% and 98.8%; and, 96.7% and 97.5%, respectively. The combined sensitivity and specificity were 100% and 96.2%, respectively.

Of interest is the SGTi-flex COVID-19 IgG rapid LFIA from Sugentec, Inc., Korea, for the qualitative detection of anti-SARS-CoV-2 IgG antibodies in human serum, plasma (sodium heparin, lithium heparin, sodium citrate, and K3-EDTA), and venous whole blood (sodium heparin, lithium heparin, sodium citrate, and K3-EDTA). The assay principle is very similar to that of Healgen's test except that recombinant SARS-CoV-2 NP and SP RBD protein is used in the conjugate. The assay takes only 10 min, and must be read visually within 30 min. Sugentec also provides a separate SGTi-flex COVID-19 IgG Control, which contains a positive and a negative control that are prepared from processed human plasma or serum. The sensitivity (PPA) and specificity (NPA) of the test to PCR were 92.43% and 99.15%, respectively. The sensitivity (PPA) was 98.6% in samples taken from COVID-19 patients ≥15 days post onset of symptoms.

The INNOVITA 2019-nCoV Ab Test (Colloidal Gold) (IgM/IgG Serum/Plasma/ Venous whole blood Combo) from Innovita (Tangshan) Biological Technology Co., Ltd., China detects and differentiates anti-SARS-CoV-2 IgM and IgG antibodies in human serum, plasma (lithium heparin, sodium citrate, and K2-EDTA), and venous whole blood (lithium heparin, sodium citrate, and K2-EDTA) [36]. The assay duration is 10 min, and results must be read visually within 15 min. The test strip has two windows, with their distinct sample wells, to selectively detect anti-SARS-CoV-2 IgM and IgG antibodies. The assay principle is similar to that of Healgen's test except that recombinant SARS-CoV-2 NP and S1 antigen is used in the conjugate. The T lines in the IgM and IgG result windows are coated with mouse anti-human monoclonal IgM (μ chain) antibodies and mouse anti-human monoclonal IgG (γ chain) antibodies, respectively. The control C lines in both IgG and IgM result

In Vitro *Diagnostics for COVID-19: State-of-the-Art, Future Directions and Role in Pandemic… DOI: http://dx.doi.org/10.5772/intechopen.97775*

windows are coated with goat anti-mouse IgG antibodies. Innovita provides separate 2019-nCoV IgM Positive Control, 2019-nCoV IgG Positive Control, and 2019-nCoV IgM/IgG Negative Control, which can be purchased separately. The positive controls are lyophilized humanized anti-SARS-CoV-2 IgM and IgG antibody in negative control serum matrix while the negative control is lyophilized negative control matrix. The PPAs to PCR for detecting anti-SARS-CoV-2 IgG and IgM antibodies in K2-EDTA plasma samples collected from COVID-19 patients ≥15 days post onset of symptoms were 97.78% and 97.67%, respectively. The NPA to PCR for the detection of anti-SARS-CoV-2 IgG and IgM antibodies was 100%. The FNLCR validation study showed sensitivity and specificity for anti-SARS-CoV-2 IgM and IgG w.r.t. the comparator method to be 93.3% and 98.8%; and, 93.3% and 98.8%, respectively. The combined sensitivity and specificity were 100% and 97.5%, respectively.

WANTAI SARS-CoV-2 Ab Rapid Test is a novel LFIA for the qualitative detection of total anti-SARS-CoV-2 Ab in human serum, venous whole blood, and plasma (K2-EDTA, sodium citrate and lithium heparin). The assay format is similar to that of Healgen test except that SARS-CoV-2 SP RBD antigens are coated at the test (T) and control (C) zones, and colloidal gold particles are conjugated to recombinant SARS-CoV-2 SP RBD antigens. The company provides separate lyophilized positive and negative controls that can be bought separately. The positive control comprises monoclonal mouse anti-SP RBD antibodies in newborn calf serum buffer. The assay duration is 15 min, and results must be read visually within 20 min. The PPA and NPA to PCR were 94.7% and 98.89%, respectively. The PPA to PCR in samples collected from COVID-19 patients ≥15 days post onset of symptoms was 91.67%. The FNLCR validation study showed sensitivity (PPA) and specificity (NPA) to be 100% and 98.8%, respectively.

#### **4. Challenges and future directions**

Various novel IVD assay formats, based on reverse transcription loop-mediated isothermal amplification (RT-LAMP), lab-on-a-chip, microfluidics, biosensor, multiplex detection, and POC technologies, are being chased by many groups. An automated, fully integrated POC COVID-19 assay, which can perform molecular, Ag, and Ab testing on a single bioanalytical platform, would be a breakthrough. The smartphone-based POC electrochemical test for SARS-CoV-2 biomarkers, similar to the iHealth Align device developed by iHealth, USA, for blood glucose monitoring [42], would be a very useful test for pandemic response. The rapid and accurate early-stage diagnosis of people infected with the SARS-CoV-2 is critical to decreasing the spread of COVID-19.

Apart from the RT-PCR based IVD tests performed in central clinical laboratories, the rapid Ag tests have played a significant role in extending the outreach of COVID-19 diagnostics to remote, decentralized, and POC settings [43]. Many well-performing rapid Ag tests have been approved by FDA under EUA and many countries for the self-testing of COVID-19. There are concerns regarding the performance of rapid Ag tests and it has been shown that they are not as accurate as RT-PCR tests. But there is no doubt that they are still very useful in pandemic response as they have enabled the mass screening of the population and helped in identifying many COVID-19 positive cases. The serology tests to detect anti-SARS-CoV-2 Ab provide the desired information to the healthcare providers about the immune status of the COVID-19 patients and convalescent subjects.

The clinical accuracy of all COVID-19 tests needs to be stringently evaluated and constantly checked. The regulatory bodies and clinical authorities should analyze if the approved IVD tests are still working properly in all the patients or they are impacted by any specific SARS-CoV-2 mutation(s). Several safety alerts have been issued by

the FDA where certain molecular tests impacted by SARS-CoV-2 mutations have been recalled from the market. Additionally, FDA has recalled a large number of COVID-19 tests due to the lacking clinical validation data or performance. The discovery and use of novel biomarkers for the diagnosis of SARS-CoV-2 infection at an early stage could further lead to a novel IVD assay that would be ideal for pandemic response.

There is a need for more extensive research so that novel rapid and highly sensitive diagnostic technologies for the POC detection of SARS-CoV-2 infection with good analytical performance could be developed. The early-stage detection of SARS-CoV-2 infection would enable the healthcare professionals to intervene early and prevent the spread of infection.

The sensitivity of rapid LFIA tests could be further enhanced by employing new nanomaterial labels [44], which could lead to much lower limit of detection for the detection of specific analytes in the patient sample. Colloidal gold nanoparticles (NPs) has been the most used in commercial LFIA rapid tests due to the availability of large number of conjugation and immobilization chemistries, easy synthesis and low cost. However, many prospective nanomaterial labels have been demonstrated to provide much higher sensitivity. These include quantum dots, up-conversion NPs, time-resolved fluorescence NPs, surface enhanced Raman scattering active NPs, magnetic NPs, carbon nanotubes and carbon NPs. In case of COVID-19, there is a need to detect very low concentrations of NP at pg/mL level, which is just at the limit of detection of conventional LFIA formats that are being used commercially in rapid tests. It is possible to increase the accuracy of detection at such low levels by employing such novel nanomaterial labels, which have been demonstrated by several researchers [44]. Several other biomarkers that are being investigated for the early-stage detection of COVID-19, such as cytokines, are also present at very low levels in the patient samples. Therefore, there is a requirement of very high sensitivity for analyte detection. Further, a large number of smartphone-based colorimetry, fluorescent and chemiluminescent readers for rapid LFIA have already been developed by many companies and groups [45–47], which would be ideal for POC readout of rapid tests and rapid transmission of test results to a dedicated healthcare server or Cloud. The current generation of smartphones have advanced imaging, processing, connectivity, and other characteristic features, which would be highly effective in the development of next generation of innovative IVD and healthcare technologies for pandemic response.
