I´m working in the development of a FPIA to detect antibodies against a viral protein of 24kDa.
Acording to the sequence, my protein has 9 lysines and 1 NH2 terminal group, so I calculate 10 tags/molecule. I have labelled the purified protein with FITC isomer 1 from SIGMA in carbonate buffer pH 9, using a FITC: protein relationship of 100:1; and then purified the conjugate by chromatography on Sephadex G25.
I ´ve made FP measurements with the Beckman DTX-880 using a buffer that is currently used for another FPA immunoassay. I obtain FPA values of 88 to 96mP with the conjugate alone, and a slight increase with positive sera (i.e. sera with antibodies to the protein, 110 to 225 mP) , but I also have a increase in mP with some negative sera (ie, sera without antibodies to the protein).
I have tested the immunological activity of the conjugated protein against my positive sera in dot blot, and the protein is still active. I need help to see what is going wrong with my FPA assay. Thank you!!
Usually FPIA is used for small molecules. The larger the analyte, the smaller the difference in the bound and the free form.
https://en.wikipedia.org/wiki/Fluorescence_polarization_immunoassay
Hello Michael, and thank you for your observation! I know FPA is ideally used for small molecules, however FPIA have been set up for antigens with MW similar or even higher tan that of my protein (flagellin protein from Leptospira of 31kDA, polysaccharide from Brucella of 22 kDa, MBP from M bovis of 18 to 23kDa...)..
Yes, you are right. However, to my knowledge, you need special labels (with long decay times) for these applications. Could you supply links to the respective articles?
Here are the links to the papers I have refered to!
http://cvi.asm.org/content/3/4/438.long
http://www.sciencedirect.com/science/article/pii/S0378113502000445?via%3Dihub
Obviously it is possible to perform FPA with antigens around 20kDa. When I checked your protocol, I was surprised about the high excess of FITC. If too many fluorphores are attached, self-quenching will occur, which might lead to strange effects. Did you check the conjugation density? About 1-2 FITC molecules per protein should be optimal. Please also consider that a high FITC load might destroy most of the epitopes, which are bound by the antibodies.
I return back to this task!!. Thank you for your answer again!!
I also think it is an excess of FITC... I did not checked the conjugation density, as I do not know exactly de extinction coefficient of my protein. Further, after some experiments I realized my protein was begining to break down, even when it was a -20°C. The antigenic properties of the protein are still OK after coupling to FITC (checked by dot and western blott).
I will make a new conjugation , adding a protease inhibitor after FITC is removed. Do you recommend me any suitable FITC/ protein relatinship, or should I have to test? (I have limited quantities of the protein!!) Is it recommended to add glycerol to the conjugate in order to preserve it at -20°C?
to increase protein stability, better add a preservative like sodium azide to prevent microbial growth and (if the assay permits) some unlabeled protein like BSA to a concentration of 5..10mg/ml, instead of costly protease inhibitors and try storage in the fridge, not in the freezer. If you suspect protease contamination in your protein prep, use a SEC column (e.g. Sephacryl S200 for your target MW range) instead of the Sephadex G25 for cleanup (the latter has a sharp cut-off at about 10kDa and just is removing unreacted fluorochrome). Contaminating proteins easily should be detectable by fluorescence, as they also will be labeled.
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