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Lateral Flow Assay Mechanism of Action Step by Step

Posted on July 22, 2021 by lateralflow 

Lateral Flow Assay, a device or strips test used to determine the presence or absence of an analyte in the sample, has a mechanism that is followed throughout the testing process. The mechanism ensures the working of the test, carrying out the procedure, and getting the results of the test. To add the fact, it must be noted that even the technology has been everywhere in the medical field and Lateral Flow Assays are not separated from that. Therefore, the technology has been incorporated into the Lateral Flow Assay but their working or mechanism remains the same. And this is one of the reasons that is the topmost testing device for developed and developing countries.

To understand the mechanism of the Lateral Flow Assay, here is a brief overview of its mechanism. First of all, there is a sample pad onto which the specimen is loaded, and that further travels to the conjugate pad. On the conjugate pad, the binding events occur due to the presence of detection particles that could be either gold or latex. The next step that occurs is that the sample and reaction agent bind and moves to the reaction membrane. The reaction membrane helps in producing results. The test line either stops or continues to flow till the adsorbent pad. To know if the Lateral Flow Assay worked the reaction agents bind at the control line and produce the results. The detailed mechanism of the Lateral Flow Assay is described next.

Sample Pad

Sample Pad is the start of the lateral flow assay test. The composition of the sample application pad is glass fiber, woven mesh, or cellulose. Glass fiber, woven mesh, and cellulose own different properties from each other. Also, the functioning and cost of them also depend on the type. Basically, the sample application pad starts the Lateral Flow Assay test and the main function of this is to get an even quantity of the specimen or the sample and distribute it. It is important for the sample pad to allow the smooth of the sample so that it can do the separation of the sample, pH adjustment, and binding, evenly. For the test to be started, the sample has to be put on the sample application pad.

Lateral-Flow-Sample-Pad
Lateral-Flow-Sample-Pad

Conjugate Pad

The conjugate pad is the second step of the Lateral Flow Assay's mechanism. Conjugate Release Pad comprises of non-woven glass fiber and there lies the detection agent which is already dried up. Linking it with the sample application pad, when the sample reaches the point of saturation and flows to the conjugate release pad and here the detection agent is released to analyze the sample. The main function of the conjugate pad is to make sure that the sample and the reaction agent are moved to the membrane while also preserving the conjugate. In the conjugate pad, there must be low non-specific binding to assure that the detection reagent is not stuck in the pad.

Lateral-Flow-Conjugate-Pad
Lateral-Flow-Conjugate-Pad

Detection Reagent

There is always the detection reagent in the lateral flow assay test. The detection reagent has antibodies that are further conjugated with the fluorescent or the colored particles. The effectiveness of the lateral flow assay is dependent on the quality of the antibodies and detection reagent.

Antibody

The selection of the antibody for the lateral flow assay test is considered important and few factors must be considered while selecting the antibody for the lateral flow assay. The first factor is to have a better supply of the antibody. Secondly, the specificity of the antigen must be checked and assured. Also, the choice between the monoclonals or polyclonals when making the choice for antibodies. Monoclonals are recommended for the fact that there could be the production of a large number of antibodies and thus helps in identifying the single antigenic epitope whereas Polycilonals can become the subject to the factor of variability and their supply is not guaranteed either.

Nitcocellulose Membrane

Substrate Membrane or Nitcocellulose Membrane is one of the most important and crucial factors in determining the Lateral Flow Assay and also towards its sensitivity. The test line and control line is drawn over the membrane to provide good binding to catch the antibodies. Non-specific adsorption on the test lines and control lines can affect the sensitivity of the results that could yield false results.

Nitrocellulose-Membrane
Nitrocellulose-Membrane

Adsorbent Pad

An adsorbent Pad or Wicking pad is positioned at the end of the strip or lateral flow assay device. The aim position of the wicking pad may have a cellulose filter that helios in optimizing the quantity of the sample in the test strip. The main function of the adsorbent pad is to control the flow of sample liquid over the nitrocellulose membrane. Not only that, but it also stops the backward flow of the sample thus maintaining the adequate quantity of the specimen or sample.

Lateral-Flow-Absorbent-Pad
Lateral-Flow-Absorbent-Pad

Process Options in Lateral Flow Assay Mechanism

Even though the step-by-step action or the mechanism has been explained in great detail, the process options in the lateral flow assay are crucial to it. There are two process options; one is batch processing and the other one is in-line/reel-to-reel assembly. Batch processing is ost-effective and needs personnel which can lead to variability in the results, thus producing non-reliable results. Whereas reel-to-reel assembly is a machine processed processing option and more reliable for larger or bulk quantities. During both types of processing options, the aspect of temperature is very important and must e considered to yield true and desirable results.

Conclusion

The Lateral Flow Assay Mechanism comprises of few parts which include pads, membranes, and some particles that help to detect the sample and produce results. Even though there are different Lateral Flow Assay devices and some of them are technologically incorporated while some are not but the mechanism remains the same. All of the factors in the mechanism of the Lateral Flow Assay must be considered important and should never be overlooked.

References
https://www.creative-diagnostics.com/Immunochromatography-guide.htm
https://fnkprddata.blob.core.windows.net/domestic/download/pdf/IBS_A_guide_to_lateral_flow_immunoassays.pdf
https://www.sciencedirect.com/topics/medicine-and-dentistry/lateral-flow-test

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