Staining with PrP Sc specific antibodies 15B3 and V5B2

One aim of the study was to examine the localisation sites of PrP^Sc. Two mAbs, 15B3 and V5B2, should be tested in immunocytochemistry (ICC) [149, 151]. These mAbs are reported to detect solely PrP^Sc without the necessity of PK digestion and GdnHCl denaturation. Thus, they could function as tools for detection of PrP^Sc without using harsh biochemical procedures such as PK digestion and GdnHCl denaturation. 15B3 bound PrP^Sc specific in

immunoprecipitation (IP), while V5B2 stained PrP^Sc selectively in immunohistochemistry (IHC) and dot blot. In a first experiment 15B3 was used in the cell ELISA assay (Figure 8). The cell ELISA protocol was not changed significantly, PK digestion remained but in one case GdnHCl denaturation was omitted (Figure 8 A, B). Uninfected N2a cells and scrapie infected H6-22L cells were stained with the same intensity, independently of GdnHCl denaturation.

Because N2a cells were identically stained as H6-22L cells, the staining appeared not to be PrP^Sc specific.

Figure 8: Conventional Cell ELISA with 15B3 for detection of PrP^Sc in scrapie infected H6-22L cells. (A) N2a cells were PK digested, not treated with 6 M GdnHCl, stained with 15B3 and gαm IgM-AP followed by a substrate incubation for alkaline phosphate. (B) H6-22L cells treated as in (A). (C) N2a cells were PK digested, incubated with 6 M GdnCHl, stained with 15B3 and gαm IgM-AP followed by a substrate incubation for alkaline phosphate. (D) H6-22L cells treated as in (C). Note that with this mAb all cell types whether scrapie infected or not showed the same staining pattern. In each picture a dark precipitate within the cells was observed.

In a next step, the cell ELISA protocol was modified for staining with mAb 15B3. Since this mAb is conformation specific, i. e. it recognises a specific conformation which is characteristic for PrP^Sc, fixation prior to staining would crosslink all proteins via aldehyde binding. This would change the conformation of all proteins. Subsequently, the staining with 15B3 should take place prior to fixation. Because PrP^Sc deposits were found within the cell (Figure 7), a mild permeabilisation step with digitonin was included. In Figure 9, N2a cells and H6-22L cells were stained with 15B3 prior to fixation. The alkaline phosphatase (AP) reaction clearly showed no difference between the two cell types. Uninfected N2a and scrapie infected H6-22L cells displayed the same staining pattern which could therefore not be due to PrP^Sc detection. Interestingly, only some cells showed a strong dark staining

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distributed over the whole cell, while in Figure 8 all cells showed a “cap-type” staining in all cells.

Figure 9: Living cells were stained according to the protocol for cell ELISA. N2a (A) and H6-22L (B) were permeabilised with 40 µg/ml digitonin for 3 min, stained with 15B3, fixed, stained with gαm IgM-AP and incubated with alkaline phosphate substrate solution. No PK digestion and GdnHCl denaturation was performed. Of note, N2a and H6-22L cells were stained with the same intensity.

Since mAb 15B3 should be used in immunofluorescence (IF) to further investigate trafficking of PrP^Sc, the cell ELISA protocol, already modified for 15B3 staining, was adapted to IF staining. N2a and H6-22L cells were permeabilised with digitonin and stained with 15B3 prior to fixation (Figure 10). Again, N2a and H6-22L cells showed staining of the same intensity. In almost all cells a so-called “cap-type” staining was visible as detected in Figure 8 for the first time.

Figure 10: Immunofluorescence staining of N2a and H6-22L cells with 15B3. N2a and H6-22L cells were permeabilised with 40 µg/ml digitonin for 3 min, incubated with 15B3, fixed, denatured with an ascending and descending sequence in 1 molar steps from 1 M to 6 M GdnHCl and incubated with gαm IgM-Cy3. Nuclei were stained with Hoechst 33342. Pictures were performed with fluorescence microscope Axiovert S100 TV, magnification 40 x, exposure time Cy3 150 ms, Hoechst 10 ms. In N2a and H6-22L cells the staining was strong.

Since uninfected N2a cells were also stained, mAb 15B3 did not detect PrP^Sc specifically.

It could be possible that 15B3 did not selectively recognise PrP^Sc in this case but also or mainly PrP^C. To exclude that 15B3 stained PrP^C, N2a cells were transfected with pCMS-PrP-EGFP 24 h prior to staining. The cells were permeabilised with digitonin and stained with 15B3 and gαm IgM-Cy3 for PrP (Figure 11). Colocalisation of Cy3 and PrP-EGFP would point to the possibility that 15B3 binds to PrP^C. However, PrP-EGFP positive cells did not show any staining with Cy3 indicating that 15B3 did not bind to PrP^C.

Figure 11: Immunofluorescence staining of Prnp transfected N2a cells with 15B3. N2a cells were transfected with pCMS-PrP-EGFP 24 h prior to staining. Cells were permeabilised with 40 µg/ml digitonin, stained with 15B3, fixed and incubated with gαm IgM-Cy3. Nuclei were stained with Hoechst 33342. Pictures were performed with fluorescence microscope Axiovert S100 TV, magnification 40 x, exposure time Cy3 40 ms, GFP 100 ms, Hoechst 10 ms. Transfected N2a cells did not show any staining with 15B3 indicating that this mAb did not specifically recognise PrP^C.

The mAb 15B3 was routinely used in IP experiments. According to these IP experiments, the mAb was applied in a buffer containing detergents. These detergents were amongst others saponin and sarkosyl which seemed to be essential for the 15B3 binding (personal communication Dr. A. Raeber and Dr. F. Kuhn, Prionics). Instead of permeabilisation with digitonin the assay was therefore adjusted using 0.5 % saponin or 0.2 % sarkosyl for permeabilisation and mAb incubation. Since living cells were used for permeabilisation, sarkosyl already lysed the cells within 2 min while saponin only permeabilised the cells.

However, both N2a and H6-22L cells showed the same staining with 15B3 indicating that saponin did not improve a specific 15B3 binding to PrP^Sc (Figure 12).

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Figure 12: Immunofluorescence staining of saponin permeabilised cells with 15B3. N2a and H6-22L cells were permeabilised with 0.5 % saponin, stained with 15B3, fixed, denatured with an ascending and descending sequence of GdnSCN in 1 molar steps from 1 M to 6 M, incubated with 2^nd Ab gαm IgM-Cy3. Nuclei were counterstained with Hoechst 33342. Pictures were performed with fluorescence microscope Axiovert S100 TV, magnification 40 x, exposure time Cy3 150 ms, Hoechst 10 ms. Changing of the permeabilisation reagent did not change the staining pattern of 15B3.

In another experiment 0.2 % sarkosyl was used for permeabilisation and mAb incubation after fixation (not shown). However, even sarkosyl did not influence the binding of 15B3 to PrP^Sc. To reduce unspecific binding of 15B3 permeabilised N2a and H6-22L cells were blocked with 10 % mouse serum, 10 % goat serum, 1 % BSA or 5 % milkpowder (MP) in PBS.

Incubation of cells with 15B3 in 1.5 % mouse or goat serum, 1 % BSA or 5 % MP/PBS followed (not shown). In this experiment the fixation of the cells was performed after primary mAb incubation. Consequently, the cells were leaking and the morphology of the cells was mostly destroyed. Unfortunately, reduction of background signals did not result in specific binding of 15B3 to PrP^Sc.

The mAb V5B2 was already successfully used in IHC of CJD infected brains. PrP^Sc deposits could be visualised with V5B2 without the use of PK and GdnHCl. The V5B2 recognises a PrP sequence, not the conformation. Thus, fixation prior to mAb incubation should not interfere with mAb binding since CJD infected brain was fixed prior to mAb incubation and staining was successful. In Figure 13, H6-22L cells were stained with 6H4 (Figure 13 A) and V5B2 (Figure 13 B). The mAb 6H4 only recognised PrP^C at the plasma membrane in the absence of GdnHCl denaturation whereas V5B2 did not stain H6-22L cells at all.

Figure 13: Immunofluorescence staining of H6-22L cells with V5B2 and 6H4. H6-22L cells were fixed, permeabilised with digitonin, stained with 6H4 (A) or V5B2 (B), followed by incubation with gαm A488. Nuclei were stained with Hoechst 33342. Pictures were taken with fluorescence microscope Axiovert S100 TV, magnification 40 x, exposure time A488 400 ms, Hoechst 20 ms. V5B2 did not stain PrP in immunofluorescence.

Several variations in the staining protocol, like permeabilisation of living cells followed by mAb incubation prior to fixation, did not lead to V5B2 binding to PrP^Sc (not shown). In addition, a dot blot was performed with cell lysates of scrapie infected H6-22L cells and uninfected N2a cells. But also in this experimental approach PrP^Sc was not stained (not shown).

5.1.3 Detection of PrP^Sc in H6-22L cells after GdnHCl denaturation by