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The do's and don'ts of Western Blot testing for EPM, and Sarcocystis sp.

S. neurona protein gel
S. neurona proteins separated on a gel and stained with Coomassie blue

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

Western Blot of S. neurona using sera from a horse with EPM
Immunobot of S. neurona probed with sera from a horse with EPM

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.

An S. neurona Western Blot using sera from an immunized mouse
S neurona antigens probed with immunized mouse sera

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.

Two dimensional WB of S neurona antigens reacted with sera from an EPM horse
Two dimensional WB of S. neurona antigens reacted with sera from a horse with EPM

When you get fancy, you can use native conditions to separate proteins first by their isoelectric point, this is a first dimension. The first dimension is then electrophoresed separating proteins by size, a second dimension. I used this technique to resolve that fat 31 kDa band into its component proteins, some were immunologically useful and some weren’t. The useful ones are detected by serum or cerebrospinal fluid from diseased horses. This is called a 2-D Western Blot.

Now the fun part, immunoprecipitation!

My next step in analysis was a technique commonly used to concentrate proteins of interest. The term for this is immunoprecipitation. Antibodies are incubated with the original, non-reduced, protein homogenate and the antibody captures interesting proteins. The antibody/protein combination is purified. This way one can define a set of proteins that would identify the host’s adaptive immunity against the parasite. I bet you can already see that comparing the serum and CSF from a diseased horse would show important differences in the immune reactions between the periphery and central nervous system!

Use the linked button to read the paper, Molecular characterization of a major 29 kDa surface antigen of Sarcocystis neurona and you can see the effect of reducing conditions, it is demonstrated in Figure 3. When you read other scientific papers that describe WB detection of antibodies in serum and CSF from infected horses, be sure to note the conditions used to prepare the blots!

Based on the science outlined above, we developed the recombinant protein for SAG 1. Recombinant proteins give us one protein in solution to continue developing a test. Not surprisingly, epitopes (host binding sites to the recombinant protein) were destroyed by sulfhydryl reduction shown in Figure 7 of the paper. Here is evidence that conformational epitopes are highly important in diagnosing host reactions to infections.

The ELISA tests that I developed use the recombinant proteins of SAG 1, 5 or 6. These proteins are three mutually exclusive, immunodominant proteins of S. neurona that infect horses. The sulfhydryl bonds are maintained when we run the ELISA. By now, you realize why it is important.

When you examine other ELISA test systems be sure and note the important factors: the protein used, if the protein is prepared under reducing or non-reducing conditions, if the protein represents the organism (by this I mean SAG 1 is different than SAG 5, and SAG 6, they are each unique, specific, and different serotypes of S. neurona). We use three different tests. If I combined the SAG’s into one large chimera, fusing the proteins together, I would lose important host-reactive epitopes that we use for case analysis.

Now it's your turn!

See if you can answer this question:

If your horse has EPM due to a SAG 1 infection, will a SAG 5 ELISA be positive or negative?

Great job if your answer was negative!

And you can move to the head of the class if you can answer this next question (you’d have to understand the last blog to get this right, go back and read it if you skipped it):

If you used proteins that were common to all Apicomplexa for your gel, would your horse be positive or negative on an ELISA if it had been exposed to S. fayeri and not S. neurona?

(Hint: S. fayeri doesn’t have SAG 1, 5, or 6 on its surface.)

Send us your questions if you have any! We are happy to explain.


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