Nitrocellulose Membrane in the Lateral Flow Assay comes after the sample and conjugate pad. Nitrocellulose membrane is also called substrate membrane and it is one of the most crucial and sensitive parts towards the results of the test conducted on the Lateral Flow Assay. In the Lateral Flow Assay, there could be different materials being used such as cellulose acetate, polyvinylidene fluoride (PVDF), charge modified nylon, and polyethersulfone (PES), etc. In the Nitrocellulose membrane, there is different pore size, porosity, thickness, and chemical treatment. Nitrocellulose membranes do not have an equal distribution of pores therefore the capillary flow time is more preferred. Before moving further into the reasoning it is important to know about the capillary flow. Basically, capillary flow time is measured in seconds or centimeters and it is the total time that the sample took to be completely filled in the test strip or the Lateral Flow Assay Device.
Nitrocellulose membrane acts as a carrier body for the antibodies on the lateral flow assay device. They are also used for the purpose of screening of different processes and types of antibodies i.e monoclonals and this occurs as the result of the modification of the enzymoimmounoassay.
On the nitrocellulose membrane, the antibodies stay and conjugate with the biological particles either latex or gold particles. The antibodies are not allowed to move and are immobilized on the nitrocellulose membrane due to various reasons. As there are test lines and control lines on the lateral flow assay, the antibodies are stuck between these two lines. Considering the test line, it is used to bind the sample protein whereas the control line has the species-specific antibodies that are used to further detect the antibodies in the sample. Another reason that the antibodies stay on the nitrocellulose membrane is the pore size of the backing film. The non-porous backing film of the nitrocellulose membrane does not allow the antibodies to slide off rather they are tightly handled and thus stay on the membrane. This also helps in reducing variability.
Other important factors that make the antibodies stay on the nitrocellulose membrane are a selection of antibody diluent, method of drying, capture antibodies’ concentration, and the rate of reagent dispensing. Proper drying is required to make sure that the capture antibodies become patched to the membrane; nevertheless, backed membranes take significantly much time to dry as compared to the unbacked membranes. Aside from the intricacy, user concerns, sample preparation, maintenance, reproducibility, and user-friendly result interpretation it should always be made sure that the sensitive detection of specified parameters. Some of these standards, such as result interpretation and sensitivity, are strongly attributable to detection agents' immobilization and spatial retention with the parameters of the testing area.
Another approach that is helpful in making antibodies stay on the nitrocellulose membrane is the covalent attachment of the antibodies to the membrane of the Lateral Flow Assay. Also, the non-covalent immobilization of the antibodies on the nitrocellulose membrane of LFA hints at the very strong capacity of the nitrocellulose membranes in binding antibodies onto it. As is aforementioned that in the Lateral Flow Assay there are particles that are sensitive and crucial to the testing of the sample. Linking this to the colloidal gold particles, they have proven to make the antigens stay on the cellulose membrane. In this process, the first step is the passive adsorption of the antibody required on the surface of the nanoparticle of gold colloidal. This is one of the most challenging in the Lateral Flow Assay. For this purpose, the conjugation particles have been created that easily make the antibodies, molecules and other elements stay on the nitrocellulose membrane.
This also offers the easy attachment and coating of the antibodies onto the surface including other analytes and even biomolecules. Other than that the latex beads in the lateral flow assay are also helpful in making antibodies stay on the nitrocellulose membrane. Also, passive conjugation can also help in making antibodies stay on the nitrocelluse membrane of the lateral flow assay test. Even though these process s and causes can make antibodies stay on the nitrocellulose membrane of the Lateral Flow Assay, they are time-taking and very long processes Not only this, but it requires large equipment and other things as well that make it hectic and very costly. Also, some causes require the active optimization of the pH and this is an extensive thing, therefore, new methodologies and processes have been incorporated into the Lateral Flow Assay device that leads to the use of other methods. A couple of antibodies are specifically immobilized on nitrocellulose by physical absorption. With deep roots in research, the antibodies can also stay on the nitrocellulose membrane due to the specific hydrophobic and dipolar interaction between both of the protein domains and nitrocellulose, which results in the preservation of orientation of antibodies.
To conclude and summarize it in light of the above description, nitrocellulose is an important part of the lateral flow assay and the device used for the testing. One of the most important and vital factors that determine the Lateral Flow Assay and its sensitivity is the Substrate Membrane or Nitcocellulose Membrane. The test and control lines are drawn over the membrane to ensure good binding and capture of the antibodies. Non-specific adsorption on the test and control lines can affect the sensitivity of the results, resulting in false result values. As nitrocellulose is one of the parts of the lateral flow assay from which the sample is passed. From the sample, the antibodies and antigenic parts of both the sample and device conjugate with each other and pass on to the next part. From there, the sample flows to the nitrocellulose membrane that consequently takes in the antibodies and antigens required for the testing. The properties and the reasons that make the stay of antigen on the nitrocellulose membrane have been described in the detail above thus covering the subject matter of the paper.
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://pubmed.ncbi.nlm.nih.gov/6350463/
https://www.bakerlab.org/wp-content/uploads/2015/12/Holstien_Anal_Bioanal_Chem_2015.pdf
https://www.nature.com/articles/s41598-021-87072-7
https://www.researchgate.net/topic/Nitrocellulose
