The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to wreak its havoc across the globe. This RNA virus is highly infectious and has infected over 188 million people worldwide, claiming more than 4 million lives. Scientists believe early detection of SARS-CoV-2 is vital as it prevents further transmission by separating healthy individuals from patients with COVID-19.
COVID-19 antibody detection
At present, real-time polymerase chain reaction (RT-PCR) assay is considered the gold standard for the diagnosis of SARS-CoV-2 infection. RT-PCR is a nuclear-derived method for detecting the presence of specific genetic material in any pathogen, including a SARS-CoV-2. Another technique used for diagnosis is serologic testing, which detects antibody dynamics developed as a response to the SARS-CoV-2 virus.
COVID-19 antibody detection in humans as an alternative diagnostic strategy has drawn a lot of attention recently. In China, several clinics have been using rapid colloidal gold, enzyme-linked immunosorbent assay (ELISA), and chemiluminescence assay approach to detect the SARS-CoV-2. However, these methods have limitations in terms of accuracy and sensitivity, with a high number of COVID-19 false positive and false negative reports observed.
A new study published in the journal Biotechnology and Applied Biochemistry deals with the development of a time-resolved fluorescence immunoassay (TRFIA) for the detection of total COVID-19 antibodies in humans. The authors of this research have stated that compared to the existing colloidal gold method and chemiluminescence method, TRFIA is a more sensitive and accurate method for the detection of the SARS-CoV-2 virus.
TRFIA: a new method for the detection of the COVID-19 virus
When infected with SARS-CoV-2, an individual produces antibodies over the course of days or weeks. The double antigen sandwich TRFIA method has been developed with a volume reaction system to detect SARS-CoV-2 specific Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies. This method is more sensitive than the double antibody sandwich method and indirect ELISA method. The TRFIA method has shown fewer false-negative results, and it can also monitor the progression of COVID-19 disease. This would help to determine patients’ responses to treatment.
The structural proteins of SARS-CoV-2, i.e., nucleocapsid (N), spike (S), envelope (E), and membrane (M), play an important role in virus replication and assembly. The S protein is the prime target of neutralizing antibodies.
In this study, a double antigen sandwich TRFIA has been optimized. The recombinant nucleocapsid phosphoprotein (N protein) and spike (S) protein of SARS-CoV-2 are immobilized on 96-well plates that can capture human COVID-19 antibodies. After the antibodies are trapped, these are then banded together with the N/S proteins labeled with europium (III) (Eu3+) chelates. Subsequently, time-resolved fluorometry was used to measure the fluorescence values. This method can determine total human antibodies which are produced after COVID-19 infection.
The authors of this study found TRFIA method had high precision and good stability. This method has shown no cross-reactivity with the negative reference of the National Reference Panel for IgM and IgG antibodies to COVID-19, and all the positive references remained positive. The authors estimated the cut-off value (CV) of the precision assay to be 3.19%. This assay was reported to be stable and could be stored for 15 days at 37°C. The optimized immunoreaction time of TRFIA is only 30 min. Another advantage of this method is that it can analyze more than 90 samples at one time. This shows that the detection time is much lower when compared to the nucleic acid detection method (RT-PCR). Both nucleic acid detection methods, as well as the ELISA method, takes around 2-3 hours for the analysis.
In order to increase the sensitivity and reduce the missing detection rate, researchers have designed simultaneous coating of SARS-CoV-2 N protein and S protein. As a result, no false positive or false negative cases were observed for the serum samples of 225 healthy individuals and 8 COVID-19 patients.
Regarding the accuracy of the TRFIA method, the researchers reported a high level of accuracy for this assay compared to other methods such as the chemiluminescence method and the colloidal gold method.
The researchers have been successful in developing an improved COVID-19 diagnosis method based on double antigen sandwich TRFIA. This technology is not only highly sensitive and specific, but it can also provide an accurate result within 30 minutes. This TRFIA method can analyze more than 90 samples (96-well plate) at a time. Hence, this detection method is appropriate for large-scale human COVID-19 total antibody testing.