Antigen tests for COVID-19

Abstract

PCR diagnosis has been considered as the gold standard for coronavirus disease 2019 (COVID-19) and other many diseases. However, there are many problems in using PCR, such as non-specific (i.e., false-positive) and false-negative amplifications, the limits of a target sample volume, deactivation of the enzymes used, complicated techniques, difficulty in designing probe sequences, and the expense. We, thus, need an alternative to PCR, for example an ultrasensitive antigen test. In the present review, we summarize the following three topics. (1) The problems of PCR are outlined. (2) The antigen tests are surveyed in the literature that was published in 2020, and their pros and cons are discussed for commercially available antigen tests. (3) Our own antigen test on the basis of an ultrasensitive enzyme-linked immunosorbent assay (ELISA) is introduced. Finally, we discuss the possibility that our antigen test by an ultrasensitive ELISA technique will become the gold standard for diagnosis of COVID-19 and other diseases.

Keywords: COVID-19; SARS-CoV-2; antigen test; real-time PCR; ultrasensitive ELISA.

Figure 1

Scheme of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This virus belongs to the genus β-coronavirus, and it is a crown-like, enveloped, non-segmented, positive-sense single-stranded RNA (+ssRNA) virus. There are four major structural proteins, including a spike surface glycoprotein, small envelope protein, membrane protein, and nucleocapsid protein.

Figure 2

Some limitations of PCR. (a) Non-specific (i.e., false-positive) amplifications are easily obtained by the effects of other viruses, bacteria, and contaminations in a laboratory. (b) The handling volume for PCR is usually a μL order, possibly failing to capture the target nucleic acids (see the left panel). If it becomes a 100 μL order or more, like that of a microplate reader well, the chance of capturing the target nucleic acids becomes larger (see the right panel).

Figure 3

Schematics of ultrasensitive enzyme-linked immunosorbent assay (ELISA) by combining sandwich ELISA with thio-NAD cycling. (a) Two antibodies used in ELISA specifically target a pathogenic protein. (b) The second antibody is labeled with alkaline phosphatase, which hydrolyzes a substrate containing phosphate. The hydrolyzed substrates are used in thio-NAD cycling. (c) Thio-NAD cycling employs 3α-hydroxysteroid dehydrogenase (3α-HSD) and its coenzymes (NADH and thio-NAD). (d) Thio-NADH accumulates in a triangular manner and can be measured at 405 nm.