Lateral Flow Assay Development Guide

Lateral Flow Assay is the device that is used for rapid testing at home and also for clinical testing. They are the most reliable and accurate devices being used in almost every developed and developing country. Also, the lateral flow Assay Development Guide has almost 10 steps to it which determine the development process of a Lateral Flow Assay. Usually, Lateral Flow Assays have a sample pad, conjugate pad, nitrocellulose membrane, labels adsorbent pad, and buffer. However, there could be some changes in the labels as there are different labels that are used in the lateral flow assays.

To start the lateral flow assay development guide, it is important to consider few actions before starting the process. First of all, it is important to analyze the format to be chosen for the lateral flow assay whether it would be competitive or sandwich format. Basically, the development guide of the Lateral flow Assay is dependent on the process of assay development. The first critical and most important step is to identify the control assay that will help to identify the target analyte in the sample. This development guide will give a general LFA development process because different assays have a different process as well.

The first step to the development guide is the selection of nanoparticles. The nanoparticles selection varies in size and shape. There are different sizes available for the lateral flow assays. There also comes the 40nm gold nanoparticles with either carbon or citrate surface that can be further used for the adsorption process of proteins. The other nanoparticles that can be selected for the development of LFA include carboxyl gold, carboxyl gold nanoshells, etc. other than that there are other probes such as cellulose beads, dyed polystyrene, and fluorescent probes. The plus point of the selection of the carboxyl gold nanoparticles is that for the adsorption process very fewer antibodies are required and due to these nanoparticles a bond is formed that is a stable and irreversible amide bond. However, it must be kept in mind that the resulting forces that are found between the nanoparticles and antibodies are due to their coupling environment.

The second step is Antibody Selection. Antibody selection is a critical step as the performance of the LFAs depends upon the affinity of the antibodies, kinetics, and other steric properties. Also, the binding of antibodies is important for the detection of the results. Another important factor while selecting the antibodies is the two types of them. The two types of antibodies are monoclonal and polyclonal. Monoclonal antibodies are easy to manufacture whereas polyclonal antibodies have relatively high sensitivity as compared to monoclonal. In the step of the antibody selection process, there are further two steps i.e. antibody purification, and control line antibody. In the antibody selection, the suggested stem is to screen the antibodies for the reason that they should be tested beforehand. The antibodies can be screened and tested by building a very preliminary and initial version of the lateral flow assay that can work as a test sample device. The performance of the antibodies is different in a different kind of LFAs. For an instance, in ELISA the kinetics are not of much importance. And talking about the lateral flow assay, there are certain conditions that antibodies must be able to do. First of all, the antibodies should be active even after they have been conjugated with the nanoparticles and their structural integrity should not be harmed in any way. Also, they must have the ability to instantly give their reaction when the sample gets rehydrated again by the sample. The incubation time in other kinds of Assays is longer as compared to the Lateral Flow Assay in which the binding to the test line must be done within the time span of few seconds.

The third process in this process is a nitrocellulose membrane and it is a very important part of the LFA as it has the test and control lines. The nitrocellulose membrane has also the reagents that work to produce the results. It is available in different sizes, porosities, and designs. Actually, the nitrocellulose membrane is dependent on the capillary flow time. By capillary flow time, it means that the time required for the sample to flow up to 4cm. In this step, there is another step that is called membrane stripping. The buffer in which a specific protein is striped can have a significant impact on the final results of any lateral flow assay. Although many proteins can be dissolved in 1x PBS, some are extremely sensitive to pH, salt concentration, and the occurrence of a stabilizer for which trehalose can be named. Membrane stripping is another important step that comes with this step. In this step, the design of the lateral flow assay test is to stripe the test and controlling the antibody lines that are present on the nitrocellulose membrane. An important consideration in this step is of nitrocellulose membrane where it is said not to keep the nitrocellulose membrane in a place where there is no moisture rather it is all dried. For the development of the lateral flow assay, the temperature for the nitrocellulose membrane should be humid. As there are different formats of assays, i.e. competitive format and sandwich format which are also very important. The antibody concentration differ in both of the formats for the sandwich format the recommended antibody concentration is 1mg/mL for only test and control line antibodies and this limit can range up to 0.5 to 2 mg/mL.  In the nitrocellulose membrane step and lateral flow development guide, another step included is the membrane blocking step. Doing this step has many benefits such as the improved flow of the fluid, test strips’ stability, and blocking the non-specific binding thus yield the true results. For most of the nitrocellulose membrane in the lateral flow assay, there is a proprietary solution that is added to the for making the membranes hydrophilic.

The next step is the conjugate and conjugate pad. For developing a lateral flow assay it is important to develop optimizing conjugating parameters. When a conjugate is prepared for the lateral flow assay it is dispensed and dried onto a conjugate pad. For developing a stabilized conjugation of antibodies there could be several steps. The steps involved in developing a cohesive and enhanced efficacy of the conjugation. In this step, conjugation stability is a very important step and therefore, colloidal stability must be optimized while designing the LFA. the colloidal stability is very essential for the lateral flow assay and therefore while designing and optimizing the LFA this is always kept in considerations. The gold nanoparticles that play a crucial role in the conjugation in LFA have very unique optical characteristics with the help of which they absorb and disperse the light with accuracy and efficacy. The strong interaction between the nanoparticles and light happens due to the conduction electrons that are present on the metal surface which result in the collective oscillation when the electrons get excited by the light with the same and equal frequency. After there is a successful conjugation there are some effects on the optical characteristics as the change is observed in the refractive index which can further be traced in the spectra of UV-vis by the distinction of the red line. Also for the treatment of the conjugation pad, there could be different methods. Some of them are immersion, or uniformly spraying with the help of an automated dispenser.

After this, the sample pad selection process occurs. This is the first part of the lateral flow assay therefore it is essential to check the sample pad material and pre-treatment of the sample pad for its flow and efficacy. The sample pads used for the lateral flow assays are of different kinds such as fiber, cotton, synthetic, etc. It has also been observed that some f the samples such as urine, blood, etc. can be varied due to many other factors which include biological factors, eating timing, drink taken before the collection of the sample on the lateral flow assay device, and many others. For treating the sample pad and optimizing it the optimized buffer can be used which has also many advantages. While developing the lateral flow assay, and selecting the sample pad the ootmized buffer can help to enhance the performance of the assay by reducing the variability of the sample that includes protein, pH, viscosity, the concentration of salt, etc. It can also improve the flow and the consistency of the assay while it’s being developed. Treatment buffers have been proved to be extremely helpful in maintaining the flow and consistency, normalizing the pH, working as blocking agents by the amalgamation of the proteins, salts, surfactants with the appropriate concentration of each.   The most important specification of the sample pad to check is the absorption capacity because that tells about the sample volume for a test.

After the sample pad is selected the wick pad selection comes next. The wicking pad helps in the adsorption process of the reagents which were not taken in by either line i.e. test line and control lines. The wicking pad also stops the backward flow of the fluid that is used as a sample. It is suggested that while selecting the material for the wicking pad the material must be checked to save the device from any discrepancies that can lead to issues and maybe false results. Other than the normal, the thick material or the wicking pad is not better than the thinner material. The thinner material is better at performing better and usually has the higher capacity material sometimes. But every material must be checked for its characteristics and their suitability for the development of the lateral flow assay. For selecting the size and material of the wicking pad it is required to keep it in alignment with the factor that the absorption capacity must be higher and buffer volume must be running.

In the lateral flow assay, the strip test assembly is also the step of its development guide. In strip test assembly, the LFAs are assembled based on large manufacturing or small-scale manufacturing. For some types of lateral flow assays, the quantitative read-out might not be the requirement but dipstick format would be required for running the strip test assembly in LFA. There is a cassette that has a strip test inside is one of the most essential and crucial steps to achieve the assay which is reproducible and also reliable. The two specifications mentioned previously are extremely significant for the quantitive tests, especially. The main work of the cassette is to provide the required pressure to the fluid. This is to ensure that there is the optimal flow and the fluid is also passing through the assembly at the same flow. The cassette also makes sure that the liquid doesn’t remainresults on the edges and just flow on the pads.

After the strip test assembly, the next step is running the assay. There are different techniques for running the assay such as district assay in the liquid conjugate, full assays for dried conjugate, and the running buffer. For the analysis of the strip, it must be known that whether the test is qualitative or quantitive and the right analysis method must be chosen. To analyze the assay, it can be either done through the naked eye in which there would be yes/no results or by capturing an impression of the test line, a flatbed scanner or a camera with controlled lighting can be used. The last step is the optimization of the assay in which choosing the right antibody pair, nitrocellulose membrane, wick pad, adsorbent pad, and all other parts are included.

Conclusion:

The development guide of the Lateral Flow Assay is nine to ten steps provided which must be followed thoroughly for creating lateral flow assay.

References

https://cdn.shopify.com/s/files/1/0257/8237/files/BioReady_Lateral_Flow_Handbook.pdf