Lateral Flow Assay Troubleshooting – Challenges with Sample Pads

March 16, 2023
By Krista Ewing, PhD, Market Manager – Diagnostics

So you’ve selected and tested your antibody and done ¾ strip testing. Everything was going well but now your sample isn’t reaching the conjugate pad. What’s next?

Is the sample pad clogged? You can try sample pads from different materials, as well as various bed volumes and thicknesses. Pads made from cellulose often have higher bed volumes and are thicker than woven meshes of glass fiber or poly-sulfones. Bonded fiber offers consistent flow and various sample filtration levels with superior strength compared to cellulose and glass fiber materials. Increasing pore size generally can help with a more challenging (i.e. viscous) matrix, but binding kinetics can suffer with increased flow speed. Extra running or washing buffer can also potentially improve the flow; however, this can dilute the target analyte. Another alternative is to utilize a combination of 2 pads to allow for a stepwise filtration approach. Having a material with a larger pore size/lower density to filter larger particles followed by a small pore size/higher density material can often help remove a wide range of particle contaminates without clogging the filtration material. Introducing a pre-filtration off-strip may be needed for certain samples to remove larger particles. This can be done using a variety of techniques, such as syringe filters or droppers with filter caps (such as Porex SQeasy).

Are the sample and conjugate pads making good contact? Ensure that sample and conjugate pads are overlaid to allow for the fluid to flow from the sample to conjugate pad. The amount of overlap can also affect the fluid flow so you may need to try varying lengths. Do you even need a sample pad or can you combine the sample and conjugate pads with one longer pad material?

Is the sample pad flooding? Perhaps the sample pad is too hydrophobic. Are the sample pads that you are testing naturally hydrophilic? If not, surfactants will be necessary to help wet the pad. Common detergents are SDS, Tween 20 and Triton. You can also increase the concentration of surfactant in the sample pad buffer to facilitate the flow of the fluid along the strip. However, be mindful of the tensile strength of your sample pad material if you plan to pre-treat using reel to reel manufacturing as the tensile strength will decrease when the material is wet. All of Porex’s LFA pad materials are made from synthetic polymer materials and do not include any glass fiber or cellulose, which ensures increased strength and durability for manufacturing.

If you didn’t see this until putting the strip into a cassette, have you checked to see if the sample pad is being compressed by the cassette? Compression on porous materials can crush the strip, altering the flow or the overlap between pad materials. Support features above the strip can ensure laminations stay in contact with overlapping materials. If your cassette has side walls, those could potentially wick the sample off the sample pad.

Looking for additional support with materials for lateral flow assay development or new sample pads to try? Reach out to our team to discuss your project and how the right materials can improve your  LFA with Porex sample pads.