Hello everyone,

I’m currently developing a lateral flow assay (LFA) to detect antibodies in serum or blood samples. For the test line, I used recombinant antigen (GST-tagged), printed using a BioDot machine. The protein was expressed in E. coli, purified, and dialyzed in 1× PBS, and tested at 1 and 2 mg/mL concentrations.

For the conjugate, I used Protein G-conjugated colloidal gold.

At this stage, I am conducting a pilot test using the dipstick method, but the ultimate goal is to develop a fully functional lateral flow assay strip.

Although I used a strongly positive serum sample, no visible test line appeared on the membrane. However, the control line consistently appears clearly, indicating the flow and conjugate are functioning properly.

To rule out issues with the serum, I performed an ELISA using the same recombinant protein, and the antigen–antibody interaction worked well in that format.

My assumption is that the recombinant protein on the membrane may not be maintaining its reactivity in the LFA format.

• What are common causes of antigen–antibody non-reactivity in LFA but not in ELISA?
• Are there any recommended additives or membrane conditions?

Any suggestions or shared experiences would be greatly appreciated.

Thank you!