Lateral Flow Assay Troubleshooting – Challenges with Conjugate Release

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

So you’ve selected and tested your antibody and your membrane, preformed your label conjugation and half strip testing. Everything was going well but now your conjugate won’t release from the conjugate pad. What’s next?

Have you tried with wet conjugate first? First be sure that your label is stable in solution after conjugation. If not, you should optimize that process and can try decreasing the buffer ionic strength, increasing surfactants, increasing the amount of bioreceptor and/or blocking with stabilizing agents (i.e. BSA) . You may need to evaluate different conjugation strategies, as the conjugation itself may not have been successful.  You then can test wet conjugate by applying mixed conjugate and sample direct to conjugate pad with no sample pad- aka ¾ strip. Is a separate blocking step needed on the conjugate pad first? Porex conjugate pads have shown excellent performance without a blocking step (below) but every assay is different.

Moving on to dried conjugate, what is your conjugate application buffer? The correct buffer is needed for stabilizing as well as re-solubilizing after dried storage. Similar to the conjugation step, stability can be affected by the ionic strength. General recommendations are to start with a low ionic strength, as the drying process creates a temporary increase in salt concentration. One option is to start with a borate buffer at 2-3mM. This will also be important when including the sample pad, as that buffer will have a key role in buffering the entire assay as the flow moves across the test strip. In addition, sugars are commonly recommended for this as they support the native conformation of dehydrated proteins. A mix of trehalose and sucrose is common. Try starting with 5% trehalose and 10% sucrose w/v, y but different variations can be tried. Increasing surfactant in the running buffer can also assist with conjugate that is stuck on the pad.

Have you tried different pad materials? Porex Conjugate Pads have demonstrated faster, complete release to nitrocellulose compared to both glass fiber and polyester conjugate pads. One assay example shown below illustrates that the faster release does not hinder the test performance, as stronger test and control line signals are seen at 10 minutes compared to glass fiber.

What is your application method? Air jet dispensing is preferred as it is more reliable, but if you are using an immersion method, how uniform is your coverage? Issues with the drying process can also create challenges with release, generating a thick solution that is unable to run through the membrane if not properly dried. Both hot air and vacuum drying have been used, although temperature above 37C should be used with caution as it could affect the stability of the bioreceptors.

If you were further on in development before you saw issues, have you considered the cassette design? If your cassette if compressing the conjugate (or sample) pad, you may see changes in the release properties as you have essentially altered the geometry of the pad by placing it under compression. Looking for additional support with materials for lateral flow assay development? Reach out to our team to discuss your project and how the right combination of reagents, bioreceptors and labels used with Porex LFA pads can improve your assay.