We are performing a microfluidics based immunoassay reaction, with a modified system. We have been getting lower readings for our spiked samples, compared to control samples. For example, if control is giving a 0.3, then sample will give 0.2, and often the value decreases further with increasing sample concentrations. We had done the assay previously multiple times, where the readings were perfect, and this is a comparatively recent phenomenon, that we have been unable to troubleshoot.
Antibodies used are monoclonal antibodies. We are binding them to the surface using a glutaraldehyde chemistry.
We are not using any HRP or other enzymatic reaction, but a proprietary detection system.
Samples used are Thyroid stimulation hormone spiked in assay buffers.
We have analysed multiple assay buffers, starting from PBS, PBS with BSA, FBS, calibrator that comes with the commercial ELISA kit, and a few others. Trend has been similar.
Blocking buffers has been mostly protein based (BSA, Casein, FBS sera, at times) with variations including Tween-20 and few other small molecules.
We are unable to understand the cause of this, and hence are asking what is the possible reason.
i suggest to contred to the bovine serum albumin (BSA) concentration before it used
This is difficult to answer without knowing more about the assay, e.g. is it a direct assay, sandwich assay, competitive assay, etc. However it is possible that there is something in your "blank" or your buffer that is binding to your antibodies, specifically or nonspecifically, and contributing to the signal, and the more of your target you include, the more you displace whatever that is, so you get an inverse response (as you might see in a competitive assay). If it's just a non-specific interaction one approach would be to hold total protein concentration constant - e.g. in your spiked samples you add your target plus BSA (or other protein), keeping the total amount of protein constant but vary the ratio of the two.
Hi Robert,
We are covalently binding the antibodies on the surface. Thus, probability of displacement is fairly low. Our buffer is mostly BSA, PBS and sometimes Tween-20.
Our assay is a sandwich immunoassay. Do you think non-specific binding can be the sole reason here?
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