The coding sequence of mature human AFU was constructed into pET22a plasmid, and the pET22a-AFU was kindly provided by Y.K. Lee’s lab in the department of Applied Chemistry, NCTU, Hsinchu, Taiwan. As previous sections, AFU was also expressed as an inclusion body from the analysis of SDS-PAGE.
To refold the misfolded AFU, the dialysis method as previously described was applied.
The pH value of the refolded protein solution was adjusted to 6 before the further purification. The pI value of AFU was 5.5, which is relatively low compared with most of the proteins. Under pH =6 AFU theoretically negatively charged, which could interact with DEAE sepharose while most of the others carried positive charge. For further purification nickel sepharose was also applied, as previous illustration (Figure 4-14).
kDa m a b c d
Figure 4-14. 12.5% SDS-PAGE analysis of protein purification from E. coli cells containing AFU: (m) protein calibration marker, (a) cell lysate of E. coli BL21 containing pET22a plasmid, (b) cell lysate of E. coli BL21 containing pET22a-AFU plasmid, (c) cell lysate of E.
coli BL21 containing pET22a-AFU plasmid under IPTG induction, and (d) refolded and
purified AFU.
Figure 4-14 shows the result of expression and purification of AFU. The E. coli cells were grown in the same conditions as described in the previous chapter. From the result of SDS PAGE analysis, AFU was expressed in the E. coli even without IPTG induction. A new protein of molecular weight about 55 kDa was presented in E. coli BL21 containing pET22a-AFU plasmid. Lane d showed the purified AFU which was further confirmed by by MALDI-TOF (Figure 4-15, and 4-16)
Figure 4-15. Protein ID confirmation by MALDI-TOF for the purified AFU: The MALDI-TOF/MS spectrum showed the peptide fragments in red matching with the partial sequence of AFU
Figure 4-16. Protein ID confirmation by MALDI-TOF for the purified AFU: The MALDI-TOF/MS spectrum showed the peptide fragments in red matching with the partial sequence of AFU
4-6. Generation of AFP-specific monoclonal antibodies
To immunize the mouse, the purified AFP-mid and AFP-tail fragments were injected to the abdomen of mouse. After injection for the three times, anti-sera were tested to verify the respective titer. The titer of anti-sera was revealed as OD405 2.3 for AFP-mid and OD405 2.5 for AFP-tail. The mouse was sacrificed for hybridoma cell production. The fused cells were separated into three 96-well plates. After 14-21 days the titer of the cell medium was tested.
According to the results of ELISA, 39 wells were selected to generate monoclonal antibodies.
The medium of each well were diluted to less than 10 cells per milliliter and distributed into a plate containing 48 wells. About 148 monoclonal cells were obtained through limiting
dilution. After 14-21 days the titer of the cell medium was tested. Only 4 clones were specifically against AFP, which were named as 3C2F4, 1D6E4, 2C3B1, and 3D8D6, respectively. Titers of the monoclonal antibodies were shown in Figure 4-17.
Figure 4-17.Titer of anti-AFP antibody. The titers of the antibodies were around 1:5000.
Western blotting was then used to identify the specificity of monoclonal antibodies against serum sample spiked with target proteins. The cell lysate from E. coli BL21cotaining pET28a plasmid and the sole serum sample were used as negative control, respectively.
0
kDa m a b c d a b c d
Figure 4-18. Identification of specificity of monoclonal antibodies 3C2F4 against serum sample spiked with AFP-mid proteins by Western blotting (right) and corresponding SDS PAGE (left). (m) prestained marker, (a) purified AFP-mid, (b) cell lysate of BL21 containing pET28a plasmid, (c) human serum, and (d) human serum spike with 1 μg AFP-mid.
kDa m a b c d a b c d
Figure 4-19. Identification of specificity of monoclonal antibodies 2C3B1 against serum sample spiked with AFP-tail proteins by Western blotting (right) and corresponding SDS PAGE (left). (m) prestained marker, (a) purified AFP-tail, (b) cell lysate of BL21 containing pET28a plasmid, (c) human serum, and (d) human serum spike with 1 μg AFP-tail.
kDa m a b c d a b c d
Figure 4-20. Identification of specificity of monoclonal antibodies 3D8D6 against serum sample spiked with AFP-mid proteins by Western blotting (right) and corresponding SDS PAGE (left), (m) prestained marker, (a) purified AFP-mid, (b) cell lysate of BL21 containing pET28a plasmid, (c) human serum, and (d) human serum spike with 1 μg AFP-mid.
kDa m a b c d a b c d
Figure 4-21. Identification of specificity of monoclonal antibodies 1D6E4 against serum sample spiked with AFP-mid proteins by Western blotting (right) and corresponding SDS PAGE (left), (m) prestained marker, (a) purified AFP-mid, (b) cell lysate of BL21 containing pET28a plasmid, (c) human serum, and (d) human serum spike with 1 μg AFP-mid.
From the result of the Western blotting Identification of specificity of monoclonal antibodies against serum sample spiked with AFP-mid proteins, 3C2F4, 1D6E4, 2C3B1, and 3D8D6 were specific to AFP. Proteins in serum and host lysate were not recognized by the antibodies.
4-7. Generation of VEGFA-specific monoclonal antibodies
Purified protein was used to immunize mice. Proteins were injected to the abdomen of mice.
After injection for three times, anti-sera were collected to test the respective titer by ELISA.
The titer of anti-sera was revealed as OD405 0.856 for VEGF. The mouse was sacrificed and the spleen was taken out for production of hybridoma cells. The fused cells were distributed
in three 96-well plates. After 14-21 days the titer of the cell medium was tested. According to the results of ELISA, 33 wells were selected to generate monoclonal antibodies. Cells of each well were diluted to less than 10 cells per milliliter and distributed to 32 wells. After 14-21 days the titer of cell medium was tested. There were only two clones producing VEGF-specific antibody, named as 3H6F8, 1E2B3. Titers of the monoclonal antibodies were shown in Figure 4-22.
Figure 4-22.Titer of VEGF specific antibodies 3H6F8 and 1E2B3. The titer of 3H6F8 was 1:3000, 1E2B3 was 1:2000.
As previously, the western blotting was then used to identify the specificity of monoclonal antibodies against serum sample spiked with target proteins. The cell lysate from E. coli
kDa m a b c d a b c d
Figure 4-23. The Identification of specificity of monoclonal antibodies 3H6F8 against serum sample spiked with VEGF proteins by Western blotting (right) and corresponding SDS PAGE (left), (m) prestained marker, (a) purified VEGF, (b) cell lysate of C43(DE3) containing pET28a plasmid, (c) human serum, and (d) human serum spike with 1 μg VEGF.
kDa m a b c d a b c d
Figure 4-24. The Identification of specificity of monoclonal antibodies 1E2B3 against serum sample spiked with VEGF proteins by Western blotting (right) and corresponding SDS PAGE (left), (m) prestained marker, (a) purified VEGF, (b) cell lysate of C43(DE3) containing pET28a plasmid, (c) human serum, and (d) human serum spike with 1 μg VEGF.
From the result of the Western blotting Identification of specificity of monoclonal antibodies against serum sample spiked with VEGF proteins, 3H6F8, and 1E2B3 were specific to VEGF. Proteins in serum and host lysate were not recognized by the antibodies.
4-8. AFU-specific monoclonal antibodies
The AFU-specific antibodies were prepared by previous work in our lab. Two monoclonal antibodies 1-52 and 3-65-3 were developed. Figure 4-25 shows the titer of these two antibodies.
Figure 4-25.Titer of AFU specific antibodies 3-65-3 and 1-52. The titer of 1-52 was 1:5000, 3-65-3 was 1:2000.
The western blotting was also used to identify the specificity of monoclonal antibodies against serum sample spiked with target proteins. The cell lysate from E. coli BL21cotaining pET28a plasmid and the sole serum sample were used as negative control, respectively.
0
kDa m a b c d a b c d
Figure 4-26.The Identification of specificity of monoclonal antibodies 1-52 against serum sample spiked with AFU proteins by Western blotting (right) and corresponding SDS PAGE (left), (m) prestained marker, (a) purified AFU, (b) cell lysate of BL21 containing pET22a plasmid, (c) human serum, and (d) human serum spike with 1 μg AFU.
kDa m a b c d a b c d
Figure 4-27. The Identification of specificity of monoclonal antibodies 3-65-3 against serum sample spiked with AFU proteins by Western blotting (right) and corresponding SDS PAGE (left), (m) prestained marker, (a) purified AFU, (b) cell lysate of BL21 containing pET22a plasmid, (c) human serum, and (d) human serum spike with 1 μg AFU.