I have been attempting this side project for a long time, and spending most of the past year troubleshooting my lateral flow test. I cannot seem to get my gold conjugates to leave the conjugate pad. Have tried 0-20% Sucrose, 0-19% BSA or glycine, and up to 2% tween-20, triton X100. Since those seem to be the most common reagents and concentrations, does anyone have any suggestions on what I could be doing wrong?
As soon as I think what I have may be "good enough", I run a dollar store test with water for comparison and realize how much my standards have been compromised. In need of serious help!!
Hii Briana Petruzzi
Can you please tell me which type conjugate release pad you r using for the LFA. I think 0-20% Sucrose, 0-19% BSA or glycine, and up to 2% tween-20, triton X100 will be suitable for the pre-treatment of the conjugate pad. But the concentration should be optimized. Do you dilute the antibody and AuNPs conjugate before loading in the release pad?
I think you have to check the Conjugate pad or the gold particles, if they o.k they released only by PBS. checked for aggregates of the gold beads.
1) Add 2.5% BSA in conjugate pad treatment to make complete release of gold
2) Use TRIS buffer pH 8.0 for your conjugate treatment as well gold diuent buffer
3) use polyester pad
4) allow your conjugate to dry atleaset 4-5 hour and prevent it from humidity
do these 4 things and u will get better gold release from conjugate pad
Pre-treat the glass fiber conjugate pads with Lateral Flow Block from www.goldcolloid.com
Rodrigo Mancini Hi, I am also developing a lateral flow assay.
I am pre-treating the conjugate pad with 0.01M PBS, 5% sucrose,1% BSA, 0.5% Tween-20 and dried it for 2 hrs at 37 degree celsius. After drying it, I immerse the conjugate pad with colloidal gold conjugates, and dry it for another 2 hours at 37 degree celsius and keep in dessicator for storage. The conjugates were able to migrate well and give signals at appropriate lines. Hope this helps.
Tan Jia Hui amazing, many thanks! And which buffer you use to run your samples? Thanks again.
Rodrigo Mancini I tested with different running buffers according to this paper and 5 mM PBS with 1% Triton X-100 worked best for me.Article Development of a Prototype Lateral Flow Immunoassay for Rapi...
Have you tried the conjugate pad pre-treatment method? Do let me know how is it.
Tan Jia Hui Hi. Thanks for your comments. How do do treat the conjugation pad? By immersing the pad in the treating solution?
Tung Minh Do Hi, if you are talking about pre-treatment, I will usually immerse the pad with the treating solution or just pipette sufficient volume of the solution to the pad as long as it is fully treated. Then I will dry it at 37 degree celsius for 2 hours before adding the antibody conjugates.
I am facing the same problem Tung Minh Do Tan Jia Hui Oded Babai . I am working of nucleic acid-based lateral flow assay to detect viruses. I am pretreating the conjugate pad with 1% BSA, 5%Sucrose, 20mM Na3PO4.12H20, 0.025% Tween-20. Can you people suggest or help me in this regard.
Hi Ahsan Ali , may I know what gold conjugates are you using and is the conjugates in good state (no aggregation)? If the gold conjugates are in good state, maybe you can try increasing the Tween-20 to 0.1% to help the release of the gold conjugates. Also, you may reduce the molarity of Na3PO4.12H2O used, perhaps 5 or 10 mM will do. Hope this helps!
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