Enzyme Linked Immunosorbent Assay (ELISA)

Finally, quantitative analytical methods that perform antigen-antibody reactions through colorimetric change with the aid of an enzyme conjugate and substrate are useful for quantitative and qualitative results in respect of molecules in biological fluids. One of the most popular of these methods is the enzyme linked immunosorbent assay (ELISA) because it can be used to quantify substances at very-low concentrations (86).

This method consists of an analytical biochemical assay with high sensitivity and specificity for the detection and qualitative or quantitative analysis of an analyte without using expensive and sophisticated devices. Any substance, whether it is a specific protein or a mixture of it, can be used as an analyte. Its methodology comprises the production of monoclonal or polyclonal antibodies using antigens. Radioimmunoassay techniques, with the use of radioisotopes or fluorescence markers, are often selected to detect proteins. In the latter method, protein quantification occurs indirectly, with the absorbance of the color generated by the chemical bond due to the the presence of the dye being proportional to the amount of protein. These techniques demonstrate good sensitivity and detection limits, and ability to quantify below the nanoscale (87). A study developed by Xiang et al. (88) reported the use of ELISA in tests for IgM and IgG antibodies directed at the diagnosis of COVID-19, obtaining strong sensitivity and specificity in relation to their detection.

However, false-negative results can occur in tests based on antibody detection. The IgM antibodies are produced as part of the early immune response during the initial stage of the infection, while the IgG antibodies indicate that the disease has entered a recovery period, or may be present if there has been prior infection (89, 90). The antibody tests are used in cases where RT-PCR is negative and there is an epidemiological bond to SARS-CoV-2 infection and during the period when symptons are first presented and the viral load is high (91). The false-negative cases, in this type of test, can occur in situations where the antibodies are close to the germline, being able to bind to the SARS- CoV-2 antigens (92). Another issue that the immunological assay presents is the high incidence of false-positive cases in seronegative patients. This is probably related to inappropriate sample collection time in relation to the stage of the infection, as well as to naive IgM antibodies, which can produce an incorrect result due to the antibodies low action. However, the search for class-switched isotypes, in this case IgG, might help to decrease the risk of errors. In addition, a target antigen is also essential when the virus being tested is capable of mutating because the same viral antigens will be present in its structure (93).

In 2004, Che et al. (37) developed and patented a group of monoclonal antibodies belonging to IgG1 or IgG2b with specific binding capacity to the SARS-CoV N protein through a hybridoma, in addition to providing the reagent for SARS-CoV antigen deletion. In a clinical application study, the double antibody sandwich ELISA kit detected the SARS-CoV antigen in the patients' serum, obtaining high specificity and sensitivity. There was no cross-reactivity with cell cultures that were not infected with SARS-CoV.

Houde and Lacroix (38) in 2004, created an in vitro diagnostic method for detecting the presence or absence of antibodies indicative of SARS-CoV by binding them to a peptide, or analog of it, to form an immune complex. In the ELISA assay the peptide was adsorbed or covalently coupled in wells of a microtiter plate treated with the serum, or the biological fluid to be tested. After washing with anti-human IgG or anti-human IgM, IgA was labeled with peroxidase and added to the wells and for peroxidase determination with a corresponding substrate. Clinical samples can range from cultured cells, cell supernatants, cell lysates, serum, plasma, biological fluid to tissue samples.

In 2005, Qin et al. (49) developed the SARS-CoV nucleocapsid protein epitope. This polypeptide has a high affinity for SARS anti-virus antibodies and the antibody developed in the invention has a high affinity for SARS-CoV. The detection method was reported to be simple, sensitive and with high precision. It uses IgG and IgM and serum or sputum samples from patients to detect the virus. Park (51) formulated a method to diagnose SARS-CoV using a nucleocapsid protein antigen (SARS-CoV-N) or spike protein (SARS-CoV-S). The method uses HRP-conjugated human anti-IgG antibody or the SARS-CoV-N monoclonal antibody mixed with a sample containing SARS-CoV to adsorb the SARS-CoV-N antibody. Finally, in 2019, Jeong et al. (61) and Han et al. (60) developed an antigen for MERS-CoV diagnosis using the NC fusion protein which included a fragment of the N-terminal domain and a fragment of the C-terminal domain of the CoV N protein. They used IgG and compared their innovations with a kit already available in the market from Euroimmun®. The invention by Han et al. showed positive results for human serum diluted 128 times, while the commercial kit showed positive results only in serum diluted 16 times. This patent used blocking ELISA, and as samples blood, body fluid, saliva, and sputum. Although both patents revealed high specificity, the latter one exhibited higher sensitivity (60).