It is early days and I am trying a very basic LFA as a proof of concept. I dispense 40 uL of a 1:5 df of stool extraction into a 4mm strip preprinted with primary Ab on test line and anti-goat IgG for control. Do I wait for the 40 uL to be completely absorbed (15min)? should the strip be dry before adding 40 uL biotinylated secondary Ab (5 ug/mL)? Should it be dry before adding 30 uL of 40 nm AuNP of 2OD? It has worked before but the test line was very faint. Is there a way to increase the protein concentration in the stool sample? If I increase dilution factor do I increase bio-secondary concentration? should I increase volumes? Frustration!
Dear Sonja,
There is a several new optimization required if you want to test the strips in real sample. Such as antibody coating concentration, GNP-Ab concentration, etc.
If you got the proof that your test line is specific then it can be optimised in real sample. Try to use sample filtration paper at sample pad to avoid non specific interaction occurred due to the proteins or metabolites present in biological sample.
Try to use different pore size membrane for the assay development.
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