Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

The overall goal of this procedure is to assess the species barrier of transmissible sponge offor encephalopathies, known as TSC to a given species without needing to challenge living animals. This is accomplished by first preparing two conversion assay substrates for use in an in vitro assay. The second step is to incubate these substrates with the TSE agents that you want to test next.

After digesting with Protase K, the samples are subjected to SDS page and immuno blooding. The final step is to measure the amount of proteinase K resistant PreOn protein in each substrate using D cytometry and compare these amounts by calculation of a conversion efficiency ratio or CER. Ultimately, the CER provides a measure of species barrier for the TSE tested to the host species from which the substrate was prepared.

Advantages of this assay include its low cost, short experimental timeframe, and replacement of living animals with tissue samples. Though this method is well suited to providing insight into the species barriers of wildlife dup prion diseases, it can be applied to any other species of interest. Generally, individuals New to this method will struggle with substrate preparation, quality control, and standardization.

After obtaining healthy uninfected brain tissue from the species of interest, prepare a 10%weight per volume homogenate in lysis buffer using doubts bead mill or mortar and pestle.Homogenization. Then further refine the homogenates by subjecting them to a syringe and needle homogenization using needles of increasing gauge until the substrate can be aspirated and expelled with ease through a 27 gauge syringe needle. Next, divide the brain homogenate into two equal volumes into separate labeled plastic conical tubes and add three molar guine hydrochloride in one XPBS at either pH 3.5 or 7.4 to each tube in a one-to-one volume ratio to brain homogenate.

Now, check the pH values of the substrate solutions and use concentrated hydrochloric acid or sodium hydroxide as needed to ensure that the pH of each substrate remains within plus or minus 0.05 of the target pH. Rotate the solutions on an end over end mixer at room temperature for five hours. Then precipitate protein by adding four volumes of methanol to the substrate solutions in each conical vortex to mix well and incubate at negative 20 degrees Celsius for 16 to 18 hours.

After the incubation sediment the samples by centrifugation at 13, 000 G for 30 minutes at four degrees Celsius. Then carefully decant and discard the S supernatants and allow methanol to evaporate from the pallets. Use cotton tipped applicators to assist in absorbing methanol clung to the inside of the tube.

Do not allow pellets to over dry. Proceed to the next step when methanol is no longer visible, use a quantity of conversion buffer equal to the amount of brain homogenate starting material to resuspend the protein pellets. After resus, briefly sonicate the substrate solutions for 10 seconds.

In a cup horn sonicate at approximately 30%of maximal power. Then aliquot the solutions into 0.5 to one milliliter volumes in labeled 1.5 milliliter micro centrifuge tubes as a cellular prion protein in both substrates should be proteinase K sensitive. Treat 10 to 25 microliters of each substrate with proteinase K at a final concentration of 100 micrograms per milliliter and digest samples at 1000 RPM at 37 degrees Celsius for one hour in the thermo shaker load the proteinase K digested substrates, along with 10 to 25 microliters of each undigested substrate.

Onto an SDS page, gel and immuno block with a prion protein specific antibody to assess host prion protein, proteinase K resistance, and levels of undigested protein. This image shows the results of the immuno blot. The first two lanes contain the undigested substrates and the two lanes on the right contain the proteinase K digested substrate.

If the protein levels in the pH 7.4 and 3.5 substrates are within approximately 10%of each other and are not proteinase K resistant, the substrate pairs are appropriate for use in CER studies store other aliquots at negative 80 degrees Celsius until ready to perform the CER assay. TSE agents should be handled using appropriate biosafety precautions and according to institutional guidelines. After obtaining TSE infected brain tissue from the species of interest, prepare a 10%weight to volume homogenate in one XPBS at pH 7.4 using the homogenization method previously used for preparation of the substrate pairs.

Aqua the resulting homogenates into 100 to 300 microliter volumes in 0.5 milliliter micro centrifuge tubes and stored at negative 80 degrees Celsius until ready to perform the CER assay. To begin slowly thaw equal amounts of the pH 3.5 and 7.4 uninfected CER substrate, pairs and TSE infected seed solutions by placing them on top of a bed of ice for approximately one hour. After thawing, keep the solutions on ice first re homogenize by aspirating and expelling each substrate solution through a 27 gauge syringe needle.

Then briefly sonicate each TSE infected solution for 10 seconds at approximately 30%of maximal power. Next, prepare conversion reactions in labeled low binding thin wall PCR tubes by pipetting 95 microliters of CER substrate. Prepared at pH 7.4 in duplicate to two of the tubes and 95 microliters of CER substrate prepared at pH 3.5 in duplicate into two further tubes.

Then add five microliters of 10%TSE infected seed solution to one tube of each pH 7.4 and 3.5 substrate. In parallel, add five microliters of conversion buffer to the other tube of each pH 7.4 and 3.5 substrate to control for non-specific non templated conversion. Also, prepare a control for input proteinase K resistant prion protein.

For each TSE agent being tested, add five microliters of 10%TSE infected seed solution to 95 microliters of conversion buffer as a control. Briefly vortex each sample in its closed PCR tube, toe and substrate. Well follow with a momentary pulse in a low speed bench top mini centrifuge to remove any volume of the reaction mixture trapped in the PCR tube lid.

Load the tubes into a bench top thermo shaker equipped to accept PCR tubes and shake samples at 1000 RPM at 37 degrees Celsius for 24 hours the following day, add sarco cell to a final concentration of 2%weight per volume and proteinase K to a final concentration of 100 micrograms per milliliter digest samples at 1000 RPM at 37 degrees Celsius for one hour in the Thermo shaker. Add SDS page sample buffer heat samples to 95 degrees Celsius for 10 minutes and resolve the remaining pronase K resistant PreOn protein in each sample by SDS page, followed by immuno blotting as before. Finally, calculate the conversion efficiency ratio as a measure of species barrier.

According to the instructions in the written protocol mouse, CER assay substrate prepared at either pH 7.4 or 3.5 was incubated with RML mouse adapted scrapy, domestic sheep, classical scrapy, white-tailed deer chronic wasting disease, or 2 63 K strain of hamster adapted scrapy. In the CER assay, the control samples labeled none contained an equal amount of infectious agent and no mouse substrate samples were analyzed for the presence of proteinase K resistant prion protein by immuno blot with monoclonal antibody. SAF 83.

Raw tric values for each sample are displayed below each lane resulting proteinase. K resistant prion protein levels were variable across mouse substrates, denatured at pH 7.4 and seated with the various TSE agents. While pronase K resistant prion protein was found in all substrates, denatured at pH 3.5 and seated with a TSE agent, this bar graph shows the average ratios plus or minus standard deviation between pH 7.4 and 3.5 mouse substrates for each infectious agent based on at least three independent assay runs.

Lowercase letters refer to statistically homogenous subsets as determined by a p value of less than 0.05 using analysis of variance. With Tookie Kramer minimum significance differences method Once mastered, this technique can be done in less than 48 hours if performed properly. The CER assay can be used as an initial assessment of the existence of A TSE species barrier that may justify or obviate additional studies in living animals.

After watching this video, you should have a good understanding of how to prepare substrates, set up conversion reactions, and calculate conversion efficiency ratios to assess species barriers.