Before getting into the working and working principles of the Lateral Flow Assay it is important to give a brief introduction about the Lateral Flow Assay. Lateral Flow Assay or to be called by another name i.e. immunochromatography is used to detect the presence or absence of the analytes to get the desired results. The main actors involved in this group are antigen, antibody species, and the specimen or the matrix. Also, Lateral Flow Assays are used for a number of detection or diagnostic purposes such as they are used for the detection of cancer by cancer marker, pregnancy, detecting drug abuse or overdose, pathogens, infectious diseases, and also for cardiac markers.
Lateral Flow Assay's working is based on 4 facets. First of all, it is a device that has a sample pad, a conjugate pad, a test line, and a control line (nitrocellulose strip). Each part of this devised is separated by a small distance that allows the smooth flow of the sample. The first step to use this device is that the sample is put on the device that is flowed to the lateral flow device fully. Once it flows the filtration process occurs where the unwanted or undesirable particulates are neutralized or filtered. Within the given duration of the Lateral flow assay test completion, the whole process occurs and thus results can be seen. When the sample reaches the conjugate pad of the device the dried particles on the pad get intermixed with the particles of the sample.
The next step that happens is the flow of analyte-bound nanoparticles and getting access to the control lines that further leads to the results. The specimen can then start flowing to the conjugate pad, which contains highly colored or fluorescent nanoparticles with an antibody on their top layer. There is also a wicking pad that absorbs the sample or specimen liquor completely to ensure that there is no liquid left behind around the test and control lines. The control line in the lateral flow device that is labeled as C is composed of affinity glands that further help to bind the nanoparticle conjugate with or without the presence of an analyte in the solution to make sure that the assay is working properly rather than the otherwise. Basically, the method of testing using this device is based on the aspects of antigen-antibody reaction. As it is above-mentioned the lateral flow assay tests can give the reading in two forms either qualitative or semi-quantitative analysis.
Leading them further to the elaborate explanation, the first stage is of the sample pad on which the sample is flowed and then absorbed. There is a chance that the filter might be on the sample pad to flow the accurate and needed amount of the sample. The second stage is on the conjugate pad. The conjugate pad starts working after the sample is absorbed and it has conjugated labels and antibodies that will calculate if the analytes are present in the sample or not for the binding events. After these two stages, the working of the nitrocellulose membrane starts working. On the nitrocellulose membrane, the analytes or the antibodies present in the sample and conjugated pad will bind together with those of the nitrocellulose pad. After this, it will pass on to the absorbent pad that will help in absorbing an extra amount of sample and producing the results.
To exemplify the working of the Lateral Flow Assay Test, the example of the antibody rapid test kit of Covid-19 can be taken. When the sample or the specimen is added to the sample pad of the lateral flow assay device it further passes through a film present on the device called filter film. Once it has been passed the presence or absence of the target analyte will be tested and if the sample will have the desirable analytes it will amalgamate with an antigen that is called colloidal-gold label antigen which will have a colored band. If there is a colored band it denotes the presence of target analytes in the specimen that was added to the Lateral flow assay test for testing and diagnostic purposes. And if there is no colored line it means the results are invalid Last but not least, there is a control line of the Lateral flow assay device which shows that enough amount of the sample has been added to the device.
To understand that how a lateral flow assay works it is also pertinent to discuss the interpretation of the results. First of all, it is important to understand the different parts of the device such as conjugate pad, sample pad, etc. So, when the conjugated antibodies gather at the control lines, the lines start to appear to show the results. The visible lines on gold nanosphere labels that are the most frequently used labels, are red in color and do not require any development. The dimensions of the gold nanoparticles determine the different shades of red, and the strength is demonstrated by the threshold of conjugated antibodies confined at the immobilized lines. If a red line appears on the test line then it indicates that the result is positive. Talking about the intensity, it depends on the amount of the analyte present in the matrix of the specimen. Also, the appearance of the control line on the lateral flow assay device is a must-have to ensure that enough amount of sample has been given and the amount is no less for testing and giving accurate results.
The working of the Lateral Flow Assay is relatively easier and understandable. Since they are not limited o the laboratories rather they work for off-laboratory purposes as well one can easily use them and understand different parts. The remarkable properties of the lateral flow assay device, and the gold nanoparticles, have made the testing an easier process and also an accessible one. They have an immense role in clinical and diagnostic purposes.