Firstly, we characterized the panel of monoclonal antibodies with ELISA to ensure that they were specific for PrPC. An ELISA plate was coated with 41 different Mabs raised against synthetic peptides of PrP. PrPknockout mouse cell lysate or wild-type mouse brain lysate was then loaded for 1 h. To detect captured PrP, biotinylated 8H4 was then incubated with the lysate and detected with HRP-conjugated strep tavidin. The majority of the antibody pairs reacted well with the BxPC3 cell lysate but not with the PrP-knockout cell lysate; 15 of them gave a high signal/background ratio (P < 0.001, and OD values for the knockout cell lysate were lower than 0.2) (Figure 1). These results therefore implied that in the ELISA reaction most of this panel of Mabs were specific for PrP.
In order to determine whether the Mabs could be used for immunofluorescence staining, we stained BxPC3 cells, a pancreatic adenocarcinoma cell line expressing PrP. PrP-knockout cells were used as a negative control. We also included a mouse IgG1 isotype control in this study. Fifteen out of the 41 antibodies tested showed good membrane staining to BxPC3 cells, but none of them reacted with PrP-knockout cells. Isotype control antibody did not react with BxPC3 cells (Figure 2). It was therefore demonstrated that some of the Mabs could be used for immunofluorescence staining of PrP.
Flow cytometry is a widely used technique for immunofluorescence labeling of proteins. It is known that not all antibodies suitable for use in immunofluoresence staining can be used successfully in flow cytometry. Of the 41 antibodies tested, 13 Mabs specifically recognized cell surface PrP, as compared to isotype control (Figure 3).
Immunoblotting is widely used to show the relative amounts and apparent molecular weights of proteins. Two Mabs, 8H4 and 8B4, have already been extensively characterized for this purpose. Here, we showed that five other Mabs could be used to detect PrP by immunoblotting, although none was as sensitive as 8H4 (Figure 4).
Finally, we investigated whether or not HPDC expressed PrPC. Using immunohistochemical staining, we have shown previously that human normal ductal cells do not express detectable PrP by (Li C, et al., 2009). PrP mRNA was not detectable using quantitative PCR (Figure 5A). Consistent with this result, no PrP staining was observed in HPDC cells, whereas PrP was clearly detectable in WV cells, a neuroblastoma cell line transfected with PrP (Figure 5B). Furthermore, immunoblotting using Mabs 8H4, 4H2, 7C11 demonstrated the presence of PrP in mouse brain lysate but not in HPDC and PrPknockout cells (Figure 5C), suggesting that normal pancreatic ductal cells in pancreas tissue probably do not express PrP, in line with our previous observations.
Figure 5. Absence of expression of PrP in HPDC cell line (PrP KO = PrP-knockout). (A) Quantitative PCR showing that the PRNP mRNA level is very low in HPDC. (B) Confocal microscopic images showing that WVT cells express PrP on the cell surface. HPDC and PrP-knockout mouse neuron cells do not express PrP. Original magnification, ×600. (C) Immunoblots showing that PrP from mouse brain has a MW of 22-31kDa, whereas there is no expression of PrP in HPDC and PrP-knockout cells. M, mouse brain; H, HPDC; P, PrP-knockout cell.
In conclusion, several Mabs raised to PrP could be used for ELISA, flow cytometry, immunofluorescence staining, and immunoblotting (Table 1). Under our test conditions, HPDC did not express PrP.
Anti-PrP Mabs 1A6 1G6 2C2 2C8 2C8.4 2D4.3 2F8 4A1 4E9 4H2 5B2 6B4 6G4 6H3 7A9 ELISA √ √ √ √ √ √ √ √ √ √ √ IFA √ √ √ √ √ √ √ FCM √ √ √ √ √ √ √ √ WB √ √ Anti-PrP Mabs 7A12 7B8 7C11 7E6 7F11 8B4 8F9.1 8G12 8H4 9H7 10G4 11G5 12A3 12A4 12H7 ELISA √ √ √ √ √ √ √ √ √ √ √ √ √ IFA √ √ √ √ √ √ √ √ FCM √ √ √ √ WB √ √ √ ? √ √ IFA: Immunofluorescent Assay; FCM: Flow Cytometry; WB: Western Blotting
Table 1. Summary of the applications of the anti-PrP Mabs