S2Sturges said:
just thought I'd throw this up here, it's the new improved EPO detection and analysis how to do sheet... Personally, I'm amazed with the amount of steps and their complexity, not to mention using a witch's brew of stuff like milk powder, mouse anti-human EPO antibody, and secondary goat anti-mouse IgG (H+L), conjugated with a horseradish peroxidase.... ?? Hard to think that a accurate and viable positive can be derived from all of this.. and so conveniently to a member of a major rival's team just before the world's biggest cycle race... I'm smelling set up...
http://www.dyeagnostics.com/site/wp-content/uploads/2015/02/Application_Note_Improvements-for-EPO-detection_Schwenke_2015.pdf
No, this is standard procedure. Milk proteins are used to reduce non-specific binding, i.e., binding of the primary antibody to proteins other than the target, in this case, EPO. The secondary antibody is used to detect the primary antibody, and thus EPO. Horseradish peroxidase, very commonly used in this procedure, is attached to the secondary antibody. It catalyzes the conversion of certain substrates to a chemiluminescent product, that is, something that emits light or glows.
Thus in a nutshell, the EPO molecules are separated by molecular weight on gels, which they move through when an electric charge is put across the gels. The gels have pores of a certain size. The smaller the EPO molecule, the more easily it passes through these pores, and thus the further down the gel it travels in a given length of time. So the molecular weight, which is used to identify the type of EPO, is inversely proportional to the distance down the gel it’s found. The EPO molecules in the gel are located by chemiluminescence, after binding to the antibodies. You actually see colored bands on the gels, and by measuring the intensity of the color, one can quantify the amount of EPO present in the band.