The Parameters to Plant Harvesting and Antibody Extraction

The Wonders to Plant Harvesting

                   Hello to all of my readers, today I would like to introduce to you all the last crucial method I have learned at the Biodesign Institute. Harvesting is a crucial method that I have used to extract the antibodies from the Nicotiana benthamiana plant leaves, which is the end result that I am working to obtain. A key point that I have to address to everyone is that harvesting involves two other techniques, which are clarification and protein A chromatography, that are important for the extraction of the antibodies from the leaves.

                Whenever I harvest a leaf, I have to first cut the leaf in half in order to remove the large, central stem vessel found on the leaf. This vessel and the plants' stems are useless to harvest, because these regions on the plants lack a suitable amount of antibodies for us to extract from; hence they have to be removed. Once a leaf is cut in half we have to remove any regions on the leaf that appear black or brownish, because these regions on a leaf indicate that there aren't a lot of antibodies that are produced. The reason why these leaves change color is either that the leaf didn't received enough water and fertilizer for it to grow healthy or it is a symptom of the leaf when it is infected by a plant bacterium, agrobacterium in this case. After all of the healthy, green leaf material has been cut off, I then have to put the leaf material into a blender in order to mash up the leaf material to form this green smoothie. The green smoothie is then filtered by using cheesecloth into four lab bottles, that are placed in a box of ice, so that the antibody proteins don't denature. What are we filtering, you may ask? We are trying to filter out the normal structural plant proteins that are found in the leaves such as rubisco for instance, so that all we have in the green smoothie are the antibodies. However, the cheesecloth isn't able to completely filter out all of the other plant proteins, enzymes, and chlorophyll pigments. That is where the two techniques, clarification and protein A chromatography, come into play.

                     The two techniques, clarification and protein A chromatography, that I mentioned above are used to extract the monoclonal antibodies from the smoothies. When I watch the graduate students perform the clarification technique, basically they continuously centrifuge the green smoothie until the green smoothie turns into a brown solution. The brown solution indicates that most of the leftover plant proteins and pigments are diminished in the smoothie, since the centripetal force produced from the centrifuge separates out these proteins and pigments from the antibodies. After the centrifuge does its job, that is when the graduate students are able to remove the separated pigments and plant proteins from the smoothie solution, which over time explains why the color of the smoothie changes color. After the graduate students finish clarification, we then move on to protein A chromatography. Protein A chromatography is basically the last filtering method that we will have to employ in order to extract the purified antibodies from this brown solution. The graduate students pour the brown solution into a lab column, where there is a filter attached and separates the divisions inside the column. Whatever solution that passes through this filter are the purified antibodies. We also have to inject a specialized affinity protein called protein A into the column. Using Protein A, which is a protein used in labs that has an affinity to an antibody's charge and attaches itself onto the antibody, the graduate students somehow were able to attach tiny iron beads to protein A. By attaching these iron beads to protein A the protein will not be able to attach to the antibodies, as these beads keep the protein A from passing through the filter. Now that this problem has been solved any solution that passes through the filter are the antibodies. The residue antibodies can then be pipetted into small lab tubes, where these tubes will hold the extracted antibodies that were able to be produced- by simple lab plants. These techniques are pretty much how we were able to extract the antibodies and show how important infiltrated plants really are in the laboratory.