ANALYSIS OF THE SELECTED CSFS

ABERDEEN TH CG McR SV5 EC SV5 SP SV5 JG SV5 SV5 = 1/400 PF2 = 1/200 MEASLES = 1/200 SV5 = 1/400 PF2 = 1/200 MEASLES = 1/200 = 1/15 = 1/15 = 1/15 = 1/15 SV5 =1/50 SV5 = 1/50 Absorption with SV5 _ FLU PBS negative results DUNDEE JL EDINBURGH DB

Table 2: Antiviral titers (anti-SV5, PF2 and anti measles) of the selected CSFs which were positive for antibodies to SV5/PF2 by indirect immunofluorescence (see table 1).

centrifugation in contrast to free antibodies which are retained in the supernatant. If purified virus is mixed with CSFs containing

antibodies to this same virus, immune complexes will result from the specific reaction of these antibodies to this virus. Because in this system, there is an excess of antigen, it is likely that, if these

antibodies are part of the CSF oligoclonal IgGs, oligoclonal bands corresponding to these antivirus antibodies will not be present in the supernatant of the CSF centrifuged after its incubation with the virus. The technique used to compare the possible difference was the silver staining IEF pattern of the supernatants of different aliquots of a CSF, each incubated with a specific purified virus or with a

negative control mixture e.g PBS.

This approach was used with the 2 selected CSFs named CG and TH. A preparation of influenza virus (a gift from the National Institute for Medical Research (NIMR)) was used as a negative control. The SV5 virus was prepared in the department (BES and B. Precious), the purity of the preparation being checked by SDS

PAGE (figure Rl-4). The respective concentration of the virus preparation was measured by dot-blot assay and the concentration of each virus each in the absorption assay was therefore standardised (1 mg/ml of virus). Five aliquots of each CSF were processed: one aliquot was incubated with purified influenza virus, one with

purified SV5 virus , one with PBS, one was not mixed with anything but left overnight at +4 °C and the last CSF aliquot kept at - 70° C overnight. Figures Rl-5a and Rl-5b show the silver staining IEF pattern of the 5 aliquots of CSF CG and CSF TH after

centrifugation. It was clear that the patterns of both CSFs were slightly different between the aliquots but it was not related to the virus itself. For example in Figure Rl-5a the patterns between aliquots did not differ greatly apart from the last track which corresponded to the CSF alone incubated at +4° C overnight. In Figure Rl-5b, however, the oligoclonal bands have almost

disappeared from CSF-TH which was incubated with PBS (track3) or with influenza (track 4). Figure Rl-6 shows another negative absorption experiment (TH repeated, EM and JL CSFs)

Different factors could have induced this effect. As previously established in the department, the technique appeared to be

reproducible and little difference was seen between different tracks of the same sample especially when they were silver stained in

Figure Rl-4: SDS-PAGE minigel of the purified SV5 preparation

used for the immunoabsorption experiments. Five pis were loaded on the gel as shown on the photograph. The proteins of the virus are named on the right side of the track.

2 3 4

Figure Rl-5a: Immunoabsorption with the CSF CG (see table 1

and 2). From left to right track 1: incubated with purified influenza virus overnight. Track 2: incubated overnight- nothing added. Track 3: incubated with SV5 overnight. Track 4: incubated with PBS

overnight. Track 5: not incubated overnight- nothing added.

2 3 4

Figure Rl-5b: Immunoabsorption with the CSF TH (see table 1

and 2). From left to right; track 1: not incubated overnight; track 2: incubated overnight with SV5; track 4 incubated overnight with influenza virus; track 5: incubated overnight.

1234 56 789 10

Figure Rl-6: Immunoabsorption experiments with 3 different

CSFs from patients with MS (JL, TH, EM). JL’s CSF and TH’s CSF are CSFs with antibodies specific for SV5 (see table 1) EM's is a CSF with no antibodies specific for SV5 (see table 1). Tracks

1,2,8,9,10: CSF from JL- track 1 and 10 incubated with PBS; track 2 and 8 incubated with measles virus; track 9 incubated with

SV5.Tracks 3 and 4: CSF from TH - track 3 incubated with SV5; track 4 incubated with PBS.Tracks 5, 6,7 : CSF from EM - track 5 incubated with measles virus; track 6 incubated with SV5; track 7 incubated with PBS.

that in the IEF of the TH and CG aliquots the tracks which were on the edges were better stained than the other tracks. This is not the case with other IEF gels of CSFs and thus the position of the tracks was not a likely explanation for the discrepancy seen between the aliquots of TH. It was then hypothesised that the ionic strength,

phosphate content or pH of the CSF mixed with different mixtures of virus or PBS may have influenced the properties of the relatively cathodic oligoclonal IgGs. It is therefore possible that non-immune reactions e.g absorption or precipitation may have been the cause of the original observations [Goswami K.K.A. et al, 1987] indicating that a large proportion of oligoclonal bands in CSFs of some MS patients were reacting to the virus SV5.

To test this hypothesis the following experiment was set up: aliquots (95 Jill) of a CSF from a patient (EM) were incubated with 5 pi of phosphate buffer solutions (phosphate 50 mM in 0.1 M NaCl) of different pH (trackl, pH 6; track 2, pH 7; track 3, pH 8). Another aliquot was incubated with phosphate buffer of pH 7 but with high salt content (0.25 M NaCl)(track 4). The last track (number 5) corresponded to the CSF alone. The aliquots were centrifuged as previously described and their supernatants run in IEF and silver stained. The result of this experiment is shown in figure Rl-7. The staining of the oligoclonal bands in the supernatant run on track 4 was less than in the other tracks. Track 5 was the aliquot which had not been diluted and the pattern was not different from track 1, 2 or 3. This correlated with previous observations made in the

department that the dilution effect due the addition of 5 pi of buffer to the 95 pi of CSF was not responsible for the difference of staining seen between aliquots which had been incubated with a virus

preparation or PBS and a CSF aliquot which has not been diluted. The overall conclusion of this experiment was that the high salt content of aliquot 4 may have induced a precipitation of some oligoclonal bands.

To corroborate this conclusion, another experiment was designed: aliquots of CSF-EM were incubated overnight with virus, PBS or on its own. They were then centrifuged and the pellet of each of these aliquots was dissolved in denaturing mixture (see Methods section, SDS-PAGE electrophoresis) and a sample run on a SDS PAGE minigel which was then silver stained. The gel is shown in Figure Rl-8. Track 1 was pellet of aliquot not diluted, track 2, 3 and

1 2 3 4 5 6

I 11

I

11

Figure Rl-7: Isoelectrofocusing of the CSF EM after changing pH

and salt content. From left to right track 1,2,3 : phosphate buffer 50 mM in 0.1 M NaCl, track 1: pH 6, track 2: pH 7, track 3: pH 8. Track 4: pH 7 in phosphate buffer 50 mM in 0,25 NaCl. Track 5: CSF incubated overnight. Track 6: CSF not incubated overnight.

Figure Rl-8: SDS-PAGE minigel of the pellets after centrifugation

of the CSF EM incubated overnight with: track 1 CSF alone, track 2 incubated with influenza virus, track 3 incubated with SV5, track 4 incubated with PBS. Heavy (H) and light (L) chains from

immunoglobulins are marked.

virus or PBS respectively. The conclusion of this experiment was that the immunoglobulins (heavy and light chain on an SDS gel) appeared in the pellet when the CSF was incubated with virus (track 2 and 3) but they were also precipitated, especially the heavy chain, when the CSF was incubated with PBS. This effect was not seen with the undiluted CSF aliquot.

In conclusion it was suggested from the above experiments that changes in ionic strength, phosphate content and pH may have been responsible for some of the differences observed in the original observation even though phosphate buffer and control antigens were run in parallel at that time.

ASSESSMENT OF THE TECHNIQUES USED IN THE