I'm trying to change the limit of detection (L.O.D) of my target analyte for an LFIA (competitive) test strip. So far, I have attempted to change both the volume of my conjugate antibodies on the conjugate pad (0.5-10 uL) and the concentrations of the antigen-BSA (50-6400 ug/mL) on the test line, some sort of a checkboard titration I would say. However, the L.O.D only seems to change slightly with a change in antibody volume/amount regardless of the change of the antigen-BSA concentration on the test line and despite the fact that my spiked sample concentration is in the low range of 30 ng/mL. My questions are:
1) Does changing the antigen-BSA concentration has any real effect on adjusting the L.O.D?
2) Is there another method for adjusting the L.O.D for competitive LFIA?
This is a complex question. However, some general considerations can be made for competitive immunoassays:
1. Lateral flow assays work with relatively high concentrations of reagents. Hence, lowering of the concentration of the reagents usually leads to higher sensitivity (in competitive assays), if the line is still detectable.
2. Since LFAs are fast assays, equilibrium cannot be assumed. This may lead to the situation that the antigen-BSA concentration does not influence the assay much.
3. The concentration of the conjugate antibodies should make a stronger effect, due to their longer contact time with the analyte.
4. Finally, many optimized competitive assays approach an affinity-limited state. This can be identified if any dilution efforts do not change the IC50 (and indirectly the LOD) anymore. In this case, a new antibody of higher affinity is required.
Thank you Michael, your answer was quite helpful. I will test monoclonal antibodies from different brands/suppliers against lower concentrations of antigen and hope to detect ones with higher affinities, as I believe an affinity-limited state has been reached.
Hi Omar. Did you determine previously the minimum quantity of antibody you need on your conjugate? Or you have used an arbitrary quantity? If by changing the amount of antibody the system has a better performance maybe you need to adjust this the first.
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