Biochemical detection reactions, such as horseradish peroxidase-based assays, surface-enhanced Raman spectroscopy (SERS), electrochemical and magnetic 2 label and detection methods have also been developed but are less commonly utilized. However, some assays have also been developed that use fluorescence detection for which a specialist reader is required. Optical detection – in particular colorimetric detection – is most frequently used as the result can be read by eye. Once the sample has been applied, the test must be left for a specified amount of time to allow the binding and subsequent detection reactions to occur.Finally, the remaining sample flows into the absorption pad at the end of the LFD.Consequently, a control line should appear even in the absence of the target analyte. This binding reaction is typically between an immobilized antibody and the antibody conjugate and is independent of the sample analyte. When the sample reaches the control line, binding occurs to indicate that conjugate release and sample transfer along the membrane has been successful.Where the target is an antibody, the test line will contain immobilized antigens that are targeted by the antibodies of interest. Where the target is an antigen, the test line will contain immobilized target-specific antibodies. This reaction is designed to be specific so that binding only occurs in the presence of the target. At the test line, binding of the conjugated target analyte (if present) to the immunoreagent in the membrane occurs. On its journey, the conjugated sample will pass through the test and control lines on the membrane. The sample continues to wick along a porous membrane, typically nitrocellulose.Where the assay is detecting antibodies, the conjugate antibody normally binds the constant region of the target antibody rather than the target-specific variable region. To prevent interference with test line binding, conjugated antibodies are designed to bind to different regions of a target antigen to the test line antibody. This is normally an antibody that will bind to the target antigen or antibody conjugated to some form of detection medium, such as colloidal gold. Here, binding occurs between the target analyte (if present) and the labeled immunoreagent. Through capillary action, the sample is then drawn along the device to the next pad, known as the conjugate pad.A couple of drops of sample is typically used. The volume must be sufficient to soak the pad and allow the sample to wick along the LFD membrane, but without flooding the device. A liquid sample is introduced to and absorbed by the sample pad located immediately beneath the sample well.Performing a direct LFT proceeds as follows (Figure 1):įigure 1: Diagram of a lateral flow device (LFD) indicating the key components and path taken by a sample. LFTs are typically housed within a plastic casing with a well for sample introduction and window through which the test and control lines can be seen. How does a lateral flow immunoassay (LFIA) work? Typically taken to refer to an LFT that detects target antibodies Typically taken to refer to an LFT that detects target antigens Table 1: Names and abbreviations used to describe LFTs.Ĭan refer to the testing process or the physical deviceĬan refer to any test that detects antigens, such as the enzyme-linked immunosorbent assay (ELISA), but includes LFTsĬan refer to any test that detects antibodies, such as the ELISA, but includes LFTsĪlthough technically not exclusively a term for an LFT and could encompass other testing modalities, this term is often taken to refer to an LFT Common terms are summarized in Table 1 below. LFTs are referred to by multiple terms and abbreviations, some of which are interchangeable, depending on whether the testing process or the device used are being referred to and whether the test detects antigens or antibodies. What are alternative common names for a lateral flow test?
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