A lateral flow assay (LFA) is a rapid diagnostic test built on the principles of immunoassay, allowing for the quick, qualitative, or semi-quantitative detection of a specific target molecule in a liquid sample. This technology, often referred to as a strip test, delivers results within minutes without requiring specialized laboratory equipment or highly trained personnel. The LFA functions essentially as a paper-based enzyme-linked immunosorbent assay (ELISA), relying on the specific binding affinity between an antibody and its corresponding antigen to produce a visible signal.
Components of the Lateral Flow Strip
The physical architecture of a lateral flow assay is an assembly of overlapping materials mounted on a plastic backing card for structural stability. Fluid movement across the strip is driven entirely by capillary action, facilitated by the porous nature of the materials. This design typically consists of four main functional sections: the sample pad, the conjugate pad, the nitrocellulose membrane, and the absorbent pad.
The sample pad, often made of cellulose or glass fiber, is where the biological specimen is applied. Its primary function is to collect the sample, filter out large particulates, and ensure a controlled flow rate into the next section. Downstream is the conjugate pad, a non-woven glass fiber matrix containing the dried detection reagent. This reagent consists of mobile antibodies specific to the target molecule, which are conjugated to reporter nanoparticles.
The reporter particles are most commonly colloidal gold nanoparticles, displaying a characteristic ruby-red color due to surface plasmon resonance (SPR). Particle size is optimized, as larger particles can increase the visual signal and assay sensitivity. The nitrocellulose membrane is the heart of the assay, displaying the binding events and results. This porous membrane is chemically treated to securely immobilize the capture antibodies at the test and control lines.
The Immunochemical Detection Process
The dynamic process begins when the liquid sample is applied to the sample pad. Capillary action then carries the fluid, rehydrating and releasing the labeled detection reagents within the conjugate pad. The released, labeled antibodies encounter the target analyte, forming a complex. The subsequent events on the nitrocellulose membrane depend on the assay format, which is determined by the size and nature of the analyte being detected.
Sandwich Assay Format
The sandwich format is employed for larger analytes, such as proteins or pathogens, that possess multiple binding sites. If the target is present, it binds to the mobile, labeled antibody from the conjugate pad. This complex then migrates to the test line (T line), which contains a second, immobilized capture antibody specific to a different epitope. The target is effectively “sandwiched” between the immobilized and labeled antibodies, causing the reporter nanoparticles to accumulate and generate a visible colored line. The intensity of this line is proportional to the concentration of the target analyte.
Competitive Assay Format
The competitive format is used for detecting small molecules, like drugs or hormones, that have only a single binding site. In this format, the test line is coated with the target analyte itself or a structural analog. If the target is present in the sample, it binds to the mobile, labeled antibody from the conjugate pad. This prevents the labeled antibody from binding to the immobilized target at the test line. Consequently, a positive result is indicated by the absence of a colored line, signifying the sample analyte successfully competed for the labeled antibodies.
Function of the Control Line
Regardless of the assay format, the control line (C line) is required to confirm that the test has functioned correctly. This line is placed further downstream from the test line and is coated with an antibody designed to capture any excess, unbound labeled antibodies or conjugates. The appearance of a colored line confirms that the sample successfully flowed the entire length of the active membrane and that the detection reagents were active. If the control line fails to appear, the test result is considered invalid.
Versatility in Diagnostics and Monitoring
The core advantages of lateral flow technology are its speed, low manufacturing cost, and robust portability, which have driven its widespread adoption in point-of-care settings globally. In human health, the technology is most recognizable in the over-the-counter home pregnancy test, which detects the hormone human chorionic gonadotropin (hCG). The LFA platform also became a staple for the rapid screening of infectious diseases, such as SARS-CoV-2 (COVID-19), influenza, and HIV, enabling decentralized testing and surveillance.
Beyond clinical medicine, the simplicity and quick turnaround of these assays have made them invaluable for applications in food safety and environmental monitoring. In food production, LFAs screen for foodborne pathogens like Salmonella, mycotoxins, and antibiotic residues in meat and milk. Furthermore, the competitive LFA format detects small-molecule contaminants, such as pesticides or heavy metal ions, in environmental samples. This allows for rapid on-site screening without the need for complex laboratory analysis.