A few years ago the NIH issued a Request for Information (RFI) regarding patient acquired samples to monitor the events prior to an autoimmune disease. Some of the papers this NIH website cited extensively analyzed cytokines but just picked a few key proteins.
Sokolove, J., Bromberg, R., Deane, K. D., Lahey, L. J., Derber, L. A., Chandra, P. E., Edison, J. D., Gilliland, W. R., Tibshirani, R. J., Norris, J. M., Holers, V. M., & Robinson, W. H. (2012). Autoantibody epitope spreading in the pre-clinical phase predicts progression to rheumatoid arthritis. PloS one, 7(5), e35296. PMC free article
Pig tissue for human autoimmune screening
A previous post on smooth muscle autoautoantibodies has covered why presenting proteins in a native conformation makes sense. Alpha-smooth muscle actin is generally considered to be the kind found in the aorta and other vascular smooth muscle. When the sequence of this 42 kDa protein is blasted against the Homo sapiens (taxid:9606) and domesticated pig Sus scrofus (taxid:9823) databases we find remarkable sequence homology. I live close to the U of A Agriculture Extension slaughter facility that sells porcine parts. The amino acid sequence of aorta (a form of smooth muscle) against the domesticated pig (Sus Scrofa) database and discovered high homology between the six isoforms of actin. Note that the human smooth muscle aorta actin has a high degree of homology to other human isoforms.
The idea is to use a “slot blot” to transfer this library of proteins to a membrane using a slot blot. After “blocking” the membrane would be probed with antibodies from the patient’s serum. A secondary antibody produced in a non human mammal that recognizes human IgG would be used to detect human IgG. This secondary antibody is conjugated with an enzyme that turns a colorless compound pink.
A. First step in greater details.
A previous post went into agonizing details as to why a digested self antigen in the periphery might look different than a digested self antigen in the thymus. A slot or dot blot is a vacuum operated device that sucks protein mixtures onto semi-porous membranes. The solutions go through, the proteins become trapped on the surface. The example in the YouTube video uses a chemical reaction that generates light. For the purpose of this proposed service, we will a color changing chemical reaction. Each dot, or slot, will contain a mixture of proteins known only to the Proteomics Core. It is very important that the proteins be in as native of a configuration as possible.
B. Second step in greater details
Say a patient reports a positive reaction with a slot (or dot) that contains a Triton insoluble fraction from pig skeletal muscle. The protein that was loaded into that particular slot will be dissolved in a much more aggressive detergent. This SDS PAGE video explains how proteins in each fraction can be separated according to molecular weight. Note that the molecular weight markers in this video are colored. Proteins in the gel are transferred to a membrane for a second round of Western blotting.
C. Third step in greater details
Ideally we will use colored molecular weight markers in the gel that will be transferred to the membrane. The mass of the marker will be plotted as a function of the distance from the top of the gel. The larger the protein, the closer to the top. Linear regression will be performed in the Kaleidegraph software to obtain an equation to calculate the mass of a band that reacts with the antibody as a function of the distance from the top of the gel. The Proteomics Core at U of A will have identified proteins in the Triton insoluble fraction from porcine skeletal muscle. The autoantigen may have a predicted mass of 105 kDa. We may have five or six proteins in this fraction that will have this predicted mass with the right post translational modification.
D. Fourth and further steps in greater details
If any of the potential matches look like they might explain the etiology of the patient’s symptoms, we can gtalk about more research lip ke assays. Drop me a line if any of this sounds intereting. We may be applying for NIH funding to develop some sort of test. Email me at Barb@BDLbiochem.com
