The do's and don'ts of Western Blot testing for EPM, and Sarcocystis sp.
If you wondered what were the initial steps to discovering what would be useful to diagnose sarcocystosis in horses you are in the right place! The early work to select an immunodominant antigen served me well for the next 22 years. Follow my steps outlined below.
Fun facts about electrophoresis
A Western Blot (WB) is an analytical molecular technique that detects proteins, separated by their size from a tissue homogenate or extract. To get useful information from a WB three things are done. Basically, a protein of interest is selected from a complex homogenate by size, the separated proteins are transferred to a support (or membrane) and then reagents like serum or specific antibodies are used to identify the protein of interest.
Generally, we are interested in proteins that have a specific size and there are fun techniques to find those interesting molecules in large mixture of proteins, the homogenate. One needs an idea of the size of the ultimate target of interest and I found my target protein for Sarcocystis neurona from the literature. The published, immunodominant protein of some pathogenic protozoa included those from mice and sheep. An important protein found in pathogenic protozoa from multiple species all were in the 30 kDa range. Proteins have an atomic mass unit, the Dalton, and k means thousand, so the most interesting size of pathogenic protozoa were 30 thousand kilodaltons.
Electrophoresis is the method used to separate proteins using a gel, the gel is like a sieve and our selection of gels changes the pore size in the molecular sieve. Proteins are charged molecules and when an electric field is applied to the protein, it moves in the electric field filtering through the sieve. You can imagine that the homogenate would have multiple proteins with similar sizes, we will get to that soon. For now, our interesting protein that would identify horses have infections with S. neurona are about 30 kDa.
When I separated my cultured Sarcocystis neurona parasites, I found through trial and error, that some buffers were better than others. A buffer is used to put the proteins in suspension to be applied to the gel. The buffer bis-tris was superior and gave me a sharp banding pattern especially in the 45kDa to 11 kDa range.
As anticipated, the homogenate contained several proteins at the 30 kDa size. In fact, 28-32 kDa proteins would confuse many EPM researchers for years to come. I needed to find the one associated specifically with S. neurona and the protein that horses reacted to when they are infected. Incubating the gel-separated proteins with serum from a horse with EPM is the tool we used to identify the important proteins. This is called an immunoblot. This immunoblot shows that there are a lot of reactive proteins at 31 kDa and 17kDa, the size proteins that captivated researchers interests. Other researchers were looking at the proteins at about 11-17 kDa proteins, I stuck with the higher banding proteins at 29-32kda. At this step the University of Florida patented the use of this protein for diagnosis of EPM but there was a lot more work to do.
The next step is immunoelectrophoresis
I immunized some rabbits and mice with the protein homogenate from S. neurona. That gave me some specific antibodies to visualize proteins of interest. Of course, a monoclonal antibody (an antibody selected to bind only one site on a protein) that would bind the protein of interest would be a prize. I compared the reactions of sera from sick horses with immune rabbit sera and found some common bands. But information was still lacking.
The issue is that homogenate preparation makes significant differences to the proteins detected with immune sera on the immunoblot. My next step was realizing that some of the detergents that are used to solubilize the organism in the homogenate decreased what I detected on my blots. The literature explained that detergents reduce the disulfide bridges in proteins. Reduced proteins adopt a random coil conformation that easily separate by size on the gel. This detergent technique is called ‘running the gel under reducing conditions’.
If there was a protein, for example 31 kDa that had two sub-units, a heterodimer, of say 15 kDa and 17 kDa, the proteins would be observed as two bands under reduced conditions. However, if the disulfide bonds were unbroken, only one 31 kDa protein would be visible.
The gel conditions change the important adaptive host responses that are detected. Detergents stretch proteins out, using detergents limit detection of the amino acids to those that sit side by side on a protein. The heterodimer described above would be lost.
By using a different protocol, I was able to separate proteins in their native condition. Amino acids that are in close proximity in a folded protein that keeps the disulfide bridging ensures the native structure that a host detects would be available for analysis. Native conformation allows proteins that are not linear to become detectable by antibodies produced when a horse is infected. Non-linear sections are called conformational epitopes.