Hello! I’ve been having trouble with designing my sandwich LFA and I’m wondering if maybe there’s a consideration related to the antibody selection process that I’m not taking into account or there is a approach to assay design troubleshooting unknown to me that I need to utilize. I began my current project without much knowledge regarding how to shop for bioreagents, making this more of a shopping challenge informed by the necessary design considerations.
During my recent efforts, I’ve attempted to identify/select a pair of antibodies through the process of elimination according to the following criteria of interest:
1. Filter settings for the initial product results: Target – my assay’s specific protein target of interest (estrogen receptor alpha), Host – Rabbit, Reactivity – Human, Clonality – Monoclonal, Conjugate – Unconjugated, No Post-Translational Modification
2. Epitopes: To make a more informed selection, I want to make sure that the antibodies I ultimately purchase “pair well” and bind to separate epitopes of my target; therefore, I select a pool of potential antibody candidates from the filtered list of products based on whether the product details specify the epitope. From what I’ve observed, certain vendors don’t often disclose the amino acid sequences of their immunogens, making this pairing aspect difficult.
3. Buffer Information: I further narrow down my list of antibody candidates to the final list of choices based on whether the concentration was provided/determined by the vendor so that I can make informed dilutions of the antibodies. I also make sure that the storage buffers are as carrier-free as possible and don’t contain anything but PBS (
As you say, there are many potential sources of difficulty with LFA. I don’t see any major issue with your thought processes on selecting suitable antibodies. However, I have a couple of observations. First, FITC is an unusual choice for LFA. How are you reading it? Often other LFA components can give high backgrounds at the wavelength required for FITC. Second, the main gap in your selection process, which is entirely understandable, is the on rate of the antibodies. Unfortunately, rarely will suppliers know this information. With lateral flow, you need a fast on rate and often you will need to screen a few pairs before finding something that works. Screening by ELISA, which many people do, is not the best approach. It is far better to screen directly by LFA. Personally I would use gold rather than FITC.
Nick Gee thanks so much for responding! As a response to your points:
1. We're using FITC because this diagnostic kit is intended to acquire quantitative results similar to a platform designed previously in my PI's lab (see attached). Our lab had designed a reader designed specifically for the task of LFA fluorescence imaging and data acquisition.
2. I definitely understand that the kinetics of antibody binding is a major factor in finding a suitable/functioning pair for a sandwich LFA; I didn't mention it in my original post, so thank you for bringing that up and reaffirming my thoughts on its importance. My selection criteria above are based on what I've deemed important amongst the product details that ARE provided by the vendors; I didn't mention kinetics in those criteria since, as you said, suppliers do not know the on rate of the antibodies.
3. I was too vague about my use of ELISA in my original post. To clarify, I've done ELISA more so as a secondary screening test to make sure that the antibody kinetics is the specific issue rather than something else. Our current screening approach has been to ONLY use ELISA after doing a preliminary screening directly by LFA (as you'd suggested) and acquiring a false negative from the test line, but maybe that's not necessary.
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