In our project, we endeavor to use AID to solve the current problems of using experimental animals and increase the availability of hybridoma technology. Although we encountered many incompatible issues during the project implementation, we still tried our best to complete our experiments. The following are our experimental results.
First, we tested the details of the lentivirus infection protocol by infecting the hybridoma with a lentivirus carrying only the GFP reporter gene. For more information about the problem we encountered, visit Engineering.
Second, we used the Western blot to confirm AID's expression and test the perfect doxycycline concentration for the Tet-On system to function in the hybridoma cell.
Third, ELISA is conducted to confirm the binding affinity of the antibody that underwent the deamination of AID against the target antigen that has surged.
Virus infection is the most crucial experiment in our entire project. To ensure that our infection protocols are correct and the target gene can be successfully expressed, we designed the lentivirus with only the GFP fluorescent gene to infect hybridoma cells as a pre-test. After the virus infection, we observed the GFP protein successfully expression in the hybridoma cells through the fluorescence microscope, confirming that our protocols are correct and the target can be successfully expressed through the lentivirus we designed. However, although GFP could be expressed in hybridoma, it greatly reduced the number of viable cell. Therefore, we designed a new construct with RFP as reporter gene. Visit Engineering to see how we tackled with the problem of toxicity of GFP.
The expression of AID plays an essential role in our project. We used lentivirus to deliver our target gene into hybridoma cells. After centrifugation and resuspension, we added tetracycline to activate the Tet-On system and expressed the downstream sequence. As (Fig. 2) shows, there is a band at 23kDa, which is the predicted molecular weight of AID confirming the successful expression of the AID protein.
In theory, adding doxycycline should induce the expression of AID, which leads to somatic hypermutation(SHM). To verify that the SHM can increase the affinity of the antibody in the hybridoma cells, we conducted the indirect ELISA to compare the antibody's binding affinity before and after AID expression.
Somatic hypermutation results in diversified antibodies. Therefore, we separated hybridoma cells into monoclone using the method shown in Design and extracted the supernatant of each well, which contained antibodies that underwent SHM to conduct the ELISA test. We coated the antigen in double dilution on the bottom of the ELISA plate. Then added the supernatant of the monoclonal hybridoma cell, which contained the target antibody. Then added the anti-mouse IgG conjugated HRP as the secondary antibody. Finally, added the TMB substrate as the coloring agent. The following figure shows that the optical density of the well below, which is the antibody that underwent SHM results from AID expression, is higher than the upper well. The ELISA's consequence confirms that our project's central concept is correct.
