Dear all,
I am working on LFA for TB diagnosis. I am also having the same issue to replicate the half-strip results in full strips where I am not obtaining test line.
Specifics of my assay
- I am using 12 um Nitrocellulose membrane from MDI; sample pad(CF3); conjugate pads(rapid 24) from Cytiva
- I am not treating the sample and conjugate pads with any buffers.
- I ain't blocking the membranes.
- I am using the same polyclonal antibodies for both capture and detection purposes.
- Conjugate ab concentration is 50 μg/ml, and test line ab conc is 0.5 mg/ml.
- I am using PBST (Tween 20-0.01%), RUNNING BUFFER and PBSTB (BSA-1%) with 5% TREHALOSE for resuspending the conjugate to 1/10 of its initial volume and thereby increasing the concentration.
- I am incubating the LFA components at 37 °C for 150 minutes before testing.
- I will apply 60 μL of sample(analyte) volume for the full strip and 10 μL of analyte solution for half-strip assays.
But every time I get a test line signal in halfstrip, but not in fullstrip.
Kindly assist me in troubleshooting the issue in the process, and provide your suggestions.
I've encountered this problem before. Typically, the antibodies in the conjugate vera denatured during drying on the conjugate substrate. Please do not use PBST as a buffer for conjugate dilution. PBS contains about 0.15 M NaCl, which causes NP aggregation during drying. Try using TRIS or Na2B4O7 instead of a PBS buffer to dilute the conjugate (5-20mm is normal). Try to increase the sugar concentration. During drying, sugars displace water molecules and help preserve the structure of the product. Try increasing the concentration to 10-20% or/and replace trehalose with sucrose. Try adding a little detergent (0.01-0.2%), eliminating BSA. Try to freeze-dry the conjugate, if possible. You can also try simply increase the concentration of conjugate. I usually dispense conjugate OD=10 1ul/mm on a conjugate pad.
If that doesn't help, just replace the antibodies. Sometimes the antibodies don't work well after drying, and that's okay.
Ok, a few ideas
1) STOP using tween 20. it is a perfectly fine supernatant, but in lateral flow it interferes with AB-Ag interaction.
2) You need to pretreat your Sample pad and conjugate pad. A PBS with 1% Casein will be ok for now, continue using trehalose
3) You probably cant change this, but polyclonal antibodies in a sandwich test is tempting fate, they are very unreliable and prone to non specific interactions.
4) Make an experiment where you add increasing ammounts of conjugate and see if you are actually using a low amount of it, is possible.
5) Is the conjugate migrating correctly?
6) you see a strange change of color of the conjugate?
7) perform a titration, is possible that your conjugate is too unstable and you need to provide better conditions, like changing the concentration of the antibody or changing the pH.
May be, some contamination or anything wrong in the kit and reagents used up. You may inform the manufacturing company and repeat the process.
Dear all,
Thanks for responding my queries in a clear manner. I have tried to incorporate most feasible suggestions. I have used Tris, HEPES buffer and have avoided PBS in the process but the issues are still present.
I was not able to respond earlier.
I have tried LOD assessment with polyclonal antibody as both test and conjugation.
I found that at higher conc there are no test zones but at lower conc there are unclear zones which I believe arises due to non specific interactions.
For negative control, there are similar irregular test zones.
in all the above experiments, I add running buffer and antigen with diluent before applying conj.
but, in one trail, I applied conj directly without any prior addition, there are no test zones.
(In the attached picture, higher conc to lower conc ranges from right to left with negative control atlast)
I found it quite tricky to solve the issue. Kindly share your views and possible suggestions by troubleshooting the assay.
Regards,
Mathan.
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