pRIA analysis consists of three phases; extraction of sample bone material from the specimen, isolation of the protein, and "solid-phase double-antibody radioimmunoassay". Typically each sample is analyzed at least 3 times.

         Extraction of the sample material consists of first removing and discarding of the outer 1-2 mm of the surfaces with a 1 mm stainless steel Dremel drill bit. Next the core bone matter is collected as chips, powder and chunks using a fresh drill bit. This material is free from significant external protein contamination and is used for the analysis.

         Isolation of the protein is performed in a 10 ml vaccutainer. The bone matter is placed into the vacutainer and capped. A solution of 1M EDTA is added via syringe through the septa. The same syringe is then used to apply a partial vacuum to the container. The bone is then dissolved in the EDTA solution for 2-5 days at room temperature while gently shaking or rotating.

         The solid-phase double-antibody radioimmunoassay is carried out in 96-well polystyrene microtiter plates. First 25 microliters of the EDTA extract in placed in the wells of the plates and allowed to sit at room temperature for 2 hours, during which time some of the protein in the solution binds to the plastic plate (thus in a "solid phase"). This is then washed out with a soy protein solution, which coats the plate to prevent any further protein binding. Next, 25 microliters of antisera raised in rabbits to albumins or sera of various species is added to the wells containing the antigen and left at room temperature overnight. During this time the antibody binds to the antigen. The more specific the antibody is to that antigen, the greater will be the binding. The plate is again washed out with soy protein and radioactive (iodine-125 labeled) antibody against rabbit gamma globulin, raised in donkeys, is applied to the wells and left overnight at room temperature. This will bind to the rabbit gamma globulin already bound to the antigen (hence, "double antibody"). The excess donkey antibody is washed out with water, and the radioactivity of the wells counted in a scintillation counter. The higher the counts, the greater the binding of specific antisera to specific antigens, and so the species of the unknown antigen can be determined.

         The results are interpreted as a "percentage" uptake of radioactivity. A known quantity of radioactivity is added to each microtiter well containing the bound sample protein. The count rates obtained in the scintillation counter are compared to the expected value of this reference material. For example, if 1000 counts were expected from pure reference material, and only 100 counts were observed from the sample, the sample would have an uptake value of 10%. This value is compared between samples to make the final determination. For example, if one antibody derived from the rabbit injections is specific to humans and another to goat, and each is injected into a microtiter well containing the bound sample protein, the one with the higher uptake percentage would indicate greater species similarity.