The ongoing spread of COVID-19 has had a devastating impact on the global population, social structure and economic growth. Rapid and accurate detection of the SARS-CoV-2 virus that causes the disease has played an important role in controlling the spread of the disease. Real-time reverse transcription PCR (RT-PCR) is the most common method used to identify SARS-CoV-2. However, this method is time-consuming and requires specialized equipment, may also result in false negatives due to low viral loads, and occasionally there is a supply chain shortage of RT-PCR reagents and consumables due to a global or localized surge in demand for the test. Therefore, countries around the world have also developed some other methods for detecting SARS-CoV-2 as a necessary supplement to the RT-PCR method. Among the diverse range of methodologies explored, lateral flow immunoassays (LFIA) stand out for their high sensitivity and specificity.
Structure of Lateral Flow Immunoassays
Nitrocellulose membrane: Acts as a flow guide and the sample flows from left to right by capillary action.
Conjugate pad: The sample is mixed with the colloidal gold-labeled antibody. If there is a target antigen in the test sample, the labeled antibody will bind to the antigen to form the labeled antigen. The conjugate pad is used to label the antigen with a colloidal gold-labeled antibody.
Detection line: Immobilises the antibody with the target antigen and detects the presence or absence of the target antigen.
Control line: Detects the validity of the colloidal gold.
How do Lateral Flow Immunoassays Work?
When the test sample (blood, urine, etc.) drops into the sample pad, due to capillary action, the sample will flow to the conjugate pad. Conjugate pads contain fluorescent (colloidal gold, etc.) labeled antibodies to the target antigen. If the target antigen is present in the sample, the labeled antibody will bind to the target antigen.
After the sample has passed the conjugate pad, it continues to travel along the device to the test line on the nitrocellulose membrane. The antigen bound to the colloidal gold-labeled antibody will bind to the immobilized antibody on the detection line, and a colored line will be formed, and the density of the line will depend on the amount of the target substance. The antigen immobilized on the detection line is called the primary antibody, which captures the target antigen.
After passing the detection line, the sample flows into the control line. The labeled antibody in the sample binds to the antibody immobilized in the control line, forming a colored line. If there is no such color band, it means that the product has failed. The antigen immobilized on the control line is called the secondary antibody, which captures the colloidal gold-labeled antibody.
If there is no target antigen in the sample, the colloidal gold-labeled antigen will not form on the binding pad, so the antibody immobilized on the detection line cannot capture the labeled antigen without developing color, but the antibody on the control line will bind to the labeled antibody and display color.
Fig. 1 The operation principle of LFIA sandwich-based method (Created with BioRender.com).
The lateral flow immunoassay (LFIA) is a popular, easy to perform and inexpensive assay for the screening, diagnosis and monitoring of COVID-19. Most of the results of LFIA are in the form of test strips, making these tests highly suitable for use by any healthcare professional or even by patients at home. Like any other testing method, LFIA has its drawbacks and false negatives and false positives are possible, but the test's rapid availability and affordability make it an effective tool for screening and detection of COVID-19 in non-clinical settings. It provides a quick and easy way to identify potential infections so that contacts of infected people can be quickly isolated and traced.
Reference
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Mirica, A.C.; et al. Latest trends in lateral flow immunoassay (LFIA) detection labels and conjugation process. Frontiers in Bioengineering and Biotechnology. 2022, 10.
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