Hi everyone,
I am working on a lateral flow rapid test using the sandwich method. I tried different parameters to get the result in the full test but no success. However, it works in the half test format.
When there is no sample and conjugate pad and I put the antigen and conjugate AuNPs in a well and run the half strip, it works and when I run the full strip it did not even show a faint line.
Any idea about what to do to solve the problem.
P.S. I want to use the strip for detecting the antigen in 100 ng/mL concentration.
the images are not very clear. Where is the test line with the half strip? Is it the colour at the edge of the half strip? If it is then this is not a signal it is the gold conjugate aggregating and not running across the strip. You may need to add or increase concentration of detergent and protein in the run buffer
It seem that the conjugate and sample pad are too long, hence the meeting point of the membrane & conugate pad must be attached very strong, put adhesive on the whole strip to allow good flow, try separate the sample pad and conjugate pad (use glass fiber for conjugate).
@Oded Babai
Thank you for the answer.
Does it make sense for the test line to disappear from the half-test to full-test?
I did all the suggestions you said, but there is no result in full strip.
@Ahmed Jehanli
The test and control line are on the top of half-strip.
sometimes when you run liquid gold conjugate by the dipstick method ( what you call half a strip) you get more marked non0specific binding than with the strip with sample pad (what you call whole strip). Can you check that the binding you see with the half strip is specific by being able to inhibit the signal with your antigen.
Hi
Do you use the same concentration and volumes of sample and conjugate for half and fullstrip? if so you should get the same results for both when you have a clean release of conjugate.
sample and conjugate pads appear too long. In general, the signal in the halfstrip is not strong enough to expect a good signal in the full strip.
It look that most of the conjugate is stuck on the pad and doesn't continue to the membrane. Try to separate the Sample application pad & conjugate pad (use glass fiber for conjugate), sorther sample pad also better, try add serfactant like triton or tween to the conjugate.
I suggest you to pre-treat the conjugation pad with one of below formula. Good luck!
A: BS (10 mM, pH: 7.4) containing: 6% trehalose, 1% (w/v) BSA, 4% sucrose, 0.05% sodium azide. B: 2 mM borate buffer, pH 7.5 containing: 1% (w/v) BSA, %0.5 tween20 and 0.05% (w/ v) sodium azide . C: 50 mM borate buffer, pH 7.5 containing: 1% (w/v) BSA, 5% trehalose [24]. D: 10 mM Tris/HCl, pH 7.4 containing: 1% (w/v) BSA, %0.5 tween20 and 1% (w/v) PEG20,000.
Ref: Optimising effective parameters to improve performance quality in lateral flow immunoassayfor detection of PBP2a in methicillin-resistant Staphylococcus aureus (MRSA)
DOI: 10.1080/17458080.2020.1775197
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