If we add equal concentrations of ConA and glucose to a dilute suspension of cit
ID: 169510 • Letter: I
Question
If we add equal concentrations of ConA and glucose to a dilute suspension of citrated red blood cells in ConA buffer, what should we expect to happen based on our data from Lab 7 (Hemagglutination Analysis of Purified ConA)? Explain why we expect to see these results. Also, if we added equal amounts of ConA and galactose to a dilute suspension of citrated red blood cells in ConA buffer, what should we expect to happen? Explain your answer. (Describe what is happening at the cellular and/or molecular level rather than what you actually see with your naked eye.)
*** Lab 7 Background Info (in case needed to answer question): Our experimental variables were ConA + mannose and ConA + galactose. In our protocol we used two 96-well plates (our ConA Dilution plate and the Reaction plate. We made dilutions of our ConA and Control ConA. We added ConA protein to A1 and A2. Then we added ConA buffer to A2 through A12. Our ConA rows were similar to Control ConA rows. In general, we tested the biological activity of our purified ConA protein.
*** Lab 7 Data: Our ConA was expected to have agglutination because we know ConA is able to bind to the sugar residues on red blood cells. In our 96-well plate we saw agglutination present in A1 through A6. However, the ConA concentration becomes so dilute that it's no longer able to agglutinate the red blood cells around A7. Similar results were seen with our ConA + galactose. With ConA + mannose there was interference by mannose so the first couple of wells have agglutination but ConA is eventually not able to agglutinate the red blood cells. The negative control had no agglutination throughout. Ultimately, we were comparing our ConA activity to the manufactured ConA activity.
In summary: Red Blood Cells + ConA ---> agglutination
* ConA = functional
RBCs + ConA:
with galactose ---> agglutination
with mannose ---> partial agglutination
Explanation / Answer
Please find the answers below:
Part 1: Addition of ConA and glucose to RBC having dilute citrate solution:
Citrate is a very well known anti-coagulant by nature. Further, the information given in the data suggests that presence of sugars is very important for clumping of RBCs by ConA treatment. This is because ConA binds to the sugars, raise the molecular weight and cause them to clump by binding to each other. It should be noted here that an antigen which binds to RBC surface is directly having a glucose moiety. Thus, addition of glucose to coagulant solution will further enhance the clumping activity of the ConA. This suggests that addition of ConA and glucose to RBC having dilute citrate solution will cause the RBCs to clump immediately.
Part 2: Addition of ConA and galactose to RBC having dilute citrate solution:
Again, it can be seen from the information given in the data that addition of ConA and galactose to RBC causes clumping by the reaction:
RBCs + ConA + galactose ---> agglutination
This is because addition of galactose further to the surface sugars of RBCs which cause them to clump.
Thus, this reaction will show immediate cellular clumping which can be visualized by naked eyes as formation of thick red clumps.