hi everybody
I want to develop a lateral flow test with the sandwich assay format. I searched for an antibody for the test line and found an antibody that has ELISA application (not lateral flow assay). can I use that as a capture antibody in the test line?
You mean fixed at the detection line? The main issues would be fixation and stabilisation of the antibody. Worth a try.
Engelbert Buxbaum
yes exactly, the antibody has to be immobilized on a nitrocellulose membrane in the test line.
Check if the antibody preparation you're intending to use has no added protein (BSA?) as stabilizer. This would reduce the amount drastically that will adsorb to the test line.
Balaji Srinivasan
thanks, Balaji.
have you ever used such an antibody for the LFA? didn't have any problem with its immobilization? or sensitivity?
Wolfgang Schechinger
many thanks, Wolfgang.
it was a good point and I had not paid attention to it.
We have used affinity purified custom antibodies from chickens (IgY) which came in bulk at 5..10mg/mL, so they were quite concentrated and did not require any stabilizers (except preservatives like sodium azide) or added proteins.
Wolfgang Schechinger
good luck. I am reviewing my protocols and I will request you if I want to use a chicken antibody.
thanks for your answer.
Balaji Srinivasan
I will definitely consider your tips in my experiments.
thank you
Falk Fish
yes, indeed, I doubt the stabilization of EIA antibodies for LFA. I have a limited budget to buy several antibodies and test them. on the other hand, I found a target antibody that is affinity-purified with the desired specifications but it has an EIA app.
You do not mention anything about your target antigen. Is it a small molecule or large, monovalent or multi-valent? Also, is the antibody polyclonal or monoclonal? With sandwich tests you you need to have a capture antibody and a detector antibody both raised to different sites of the molecule. Best example is the pregnancy lateral flow test where one use anti-HCG beta chain antibody as capture and anti- alpha HCG alpha chain antibody for detection. It is possible to use the same antibody for both capture and detection if the target antigen is a large molecule with several epitopes and your antibody is polyclonal. A monoclonal antibody (which will only bind to one eptitope) can be used for both detection and capture if your antigen is multi-valent ( for example has two identical chains)