The sample is applied to the sample pad and flows to the conjugate pad where it resolubilizes the antibody gold nanoparticle conjugates which recognise the analyte. The sample with the antibody gold nanoparticle conjugates flows towards the test line on the nitrocellulose membrane through capillary force. At the test line a signal is generated depending on the concentration of the analyte. At the control line, a signal appears as the immobilised secondary antibody binds the antibody on the gold nanoparticle conjugate, indicating a successful test. The absorbent pad absorbs the liquid and ensures a continuous flow.
For SARS-CoV-2 rapid antigen tests and pregnancy tests, the sandwich assay is used as test format because coronavirus and human chorionic gonadotropin (HCG) are large analytes. A specific antibody for the analyte is immobilised on the test line leading to the analyte being caught in a ‘sandwich’ between the antibody on the test line (capture antibody) and the antibody on the gold nanoparticle (detection antibody). Gold nanoparticles have an intense red colour and are therefore well suited as a label in lateral flow assays. In sandwich assays, the signal intensity on the test line increases with the analyte concentration.
The sandwich assay is not suitable for the detection of small molecules (< 1000 Da), as it is sterically impossible for two antibodies to bind to a small molecule simultaneously. Therefore, an alternative assay format is needed. The analyte (derivative) is immobilised on the test line. The detection antibody can only bind to the test line if it has free paratopes. Since in this test format the analyte in the sample competes with the analyte (derivative) on the test line for the paratopes of the detection antibody, it is called a competitive assay.
