The purpose of this lab was to test the biological activity of ConA by performing a hemagglutination assay. If ConA is active then agglutination will occur due to ConA’s free receptors being able to bind to the glucose residues on the sheep’s red blood cells. If ConA is not active then no agglutination will occur. To test the hemagglutination reaction, two types of ConA solutions were compared, a purchased control ConA solution in buffer as the positive control, and a purified solution of ConA in buffer previously purified in lab. Each solution was at a 2mg/ml concentration of ConA in ConA buffer, which is necessary for ConA’s biological activity. Two variables were added, Galactose and Mannose, to the ConA solution to compare the effects each had on the hemagglutination reaction. I hypothesize for ConA to be able to agglutinate the red blood cells if in the adequate concentration and if in the presence of Galactose, not Mannose. Mannose will inhibit the ConA from binding to the red blood cell’s membrane, preventing agglutination. RESULTS
The reaction plate containing the ConA dilutions was incubated over the weekend and resulted in all wells being pink, appearing as if every well had agglutinated. There was a vague outline of the non-agglutinated cells in various wells. The last agglutination was observed at titer 0.0625 (1/16). Agglutination was seen in rows A, B D, and E (row A contained the control ConA, row B contained the control ConA + Galactose, row D contained the sample ConA, and row E contained the sample ConA + Galactose). In the well rows C and F which contained control ConA + Mannose and sample ConA + Mannose, agglutination did not occur at any concentration of ConA. Row G, the negative control appeared to have agglutinated as well as Row H, which contained only ConA buffer.
DISCUSSION AND CONCLUSION
The results did not support my hypothesis for the biological activity of ConA. There are some sources of error that could explain the results obtained. It’s possible there was a problem with either the ConA buffer or the sheep red blood cells to allow for all wells to turn pink and appear agglutinated. Another explanation of the irregular results was there might have been cross contamination from not changing tips when transferring to different ConA concentrations, or if bubbles were introduced while diluting the ConA, making the results difficult to interpret.
For wells A, B D, and E as ConA became more diluted or decreased in concentration, it became more difficult for it to effectively crosslink and agglutinate the red blood cells. Well D, the positive control that contained the purchased ConA resulted in agglutination of the first couple wells, then no agglutination as the ConA concentration decreased, similar to Row A. Wells B and E that had the Galacatose additive obtained the same titer of the control ConA because ConA does not bind Galactose. Galactose doesn’t interfere with ConA from binding to the sugar residues on the red blood cells. Mannose on the other hand, is an inhibitor to ConA’s binding sites. The Mannose in solution competed with the ConA and did not allow to bind to the sugar residues on the red blood cells as seen in rows C and F. Row G, the negative control, should have resulted in non-agglutination, similar to the rows containing the Mannose additive. The results observed showed agglutination formed in this row. Lastly, Row H should have shown non-agglutination through out because the well contained only ConA buffer, not ConA protein.
In conclusion, the results did not clearly explain the biological activity of ConA with the hemagglutination assay. The experiment contained too many anomalies to get a clear determination of ConA’s functionality post purification. The results did show that a change in the concentration of ConA would alter the strength of the reaction. Also, ConA’s ability to bind to sugar residues can be affected if ConA has to compete or is inhibited to bind to a cells membrane. LITERATURE CITED
Cell Biology 3822 Lab Manual, Cell Surface Glycoprotein Receptor Analysis
Using Concanavalin A Lab 7. Pearson Learning Solutions. 2012: 147-154.
Madeleine Zaechringer. Cell Biology 3822 Analysis of purified ConA via Hemagglutinatino Assay Lab 7: Powerpoint. 2014.