An In Vitro Diagnostic for Multiple Sclerosis Based on C-peptide Binding to Erythrocytes
Access original Article: http://www.ebiomedicine.com/article/S2352-3964(16)30348-6/fulltext
Publish Date: August 4, 2016
Significance: The startup company that I’ve been working for upon graduating from Michigan State University last December, is being spun out of my advisor, Dr. Dana Spence’s lab. We have a diagnosis for multiple sclerosis (MS) from a simple blood draw with a sensitivity of 98% and a specificity of 90%. Of note, we do not foresee our test replacing the current process, which is a diagnosis through attrition, but working in concert with existing procedures to aid doctor’s during their decision making. What makes our diagnostic so straight forward is the fact that our method lies in the sample prep as we use ELISA kits that are already commercially available, and approved by the Food and Drug Association (FDA). This paper was our first official publication, in which we communication the to scientific community exactly what we have discovered. In short, we applied our diagnostic to a 200 patient preliminary study, in which the patients were healthy, had either MS or other neurological diseases (OND), e.g., NMO, Parkinson’s, migraines, epilepsy. Our sample preparation involves isolating and washing erythrocytes or more commonly known as red blood cells (RBCs) before incubating them with C-peptide. Our test measured the uptake of C-peptide by the RBCs, with MS patents exhibiting significantly higher levels of C-peptide uptake in comparison to OND patients or healthy individuals.
Future Studies: The next step for us is to prepare for an actual Clinical Study through the FDA. We are very excited for the potential applications for this diagnostic in the context of a reduced diagnosis time for patients that way they can get on the approve therapies sooner! We have many other studies we plan on eventually running, especially in context to how early our test will work? Perhaps we can diagnosis MS prior to any symptoms presenting? Again, we don’t know, however we are eager to start such studies.
C-peptide: The C-peptide molecule is essentially a byproduct of insulin formation by the beta cells in the pancreas. A larger molecule, proinsulin, upon being cleaved, yields one unit of insulin and one of c-peptide. Traditionally, C-peptide is viewed as only functioning to arrange insulin in the correct shape, however there are various scientific groups that believe this 31 amino acid peptide has other biological activity. You will have probably heard of C-peptide in the context of a test that newly diagnosed type 2 diabetes patients take to in monitor insulin levels, since the two molecules are produced in equal quantities.
Enzyme linked immunosorbent assay (ELISA): ELISAs, specifically sandwich type, are the tests we use to actually measure the quantity of C-peptide in our solution. The assay comes in plate format, with each plate containing 96 wells, each of which are coated with an antibody amenable to C-peptide. Therefore, once we open the plate, we add our prepared samples, each to their own well and wait an allotted amount of time for the peptide to bind to the antibody on the plate. That way, when washed the C-peptide will remain behind in the plates for further analysis. After washing, a second antibody, the substrate is added which also binds to the C-peptide. The purpose of the second antibody is to bind the substrate, which will turn color in response. Therefore, the higher concentration of C-peptide in the well will result in a more concentrated color being present. A ‘stop’ solution is then added to halt the color change from continuing to develop to allow for the plate to be analyzed using a spectrometer.
Red blood cells (RBC): RBCs, i.e., erythrocytes, are found in your bloodstream and are most commonly associated with the delivery of oxygen throughout the body. RBCs do not contain a nucleus and are a concave disc shape that is approximately 8 micrometers in diameter. The average RBC has the average lifespan of 100-120 days, with an average human body containing 20-30 trillion cells.
Multiple sclerosis (MS): MS, a disease that attacks the central nervous system (CNS), affects approximately 2.5 million people worldwide. The diagnosis process is one of attrition, often taking months, if not years to officially diagnose. It is well established in scientific literature that the earlier patients get on disease modifying therapies for MS that their disease progression will have a higher probability of slowing or maintaining. The McDonald Criteria is used to diagnose patients that have MS, based on a set of diagnostic criteria that monitor CNS lesions, i.e., damage to the CNS, that are disseminated in space and time using MRIs or spinal taps. Another way of explaining would be that the damage is observed on different dates and in different areas of the CNS. While this is a bare bones description of the McDonald’s Criteria, you can see a more detailed table here.
Sensitivity and Specificity: A simple definition of sensitivity is the ability of the diagnostic to correctly diagnose patients that have a disease (true positive). A simple definition of specificity is the ability of the diagnostic to correctly separate out the patients that do not have the disease (true negative). Therefore, a cutoff point for the diagnostic is set on a defined value and based on how the patients fall on this scale will yield a tests sensitivity and specificity. Therefore, if someone that has the disease tests negative, it will lower the tests specificity (false negative), and if someone that does not have the disease tests positive on the scale, it will lower the sensitivity (false positive).