The effect of the absence of T and B-cell responses, and consequently antibody, on the removal of chromium labelled erythrocytes was primarily studied to gain some insight into the involvement of antibody in the premature removal of uninfected RBCs. The results indicated that uninfected erythrocytes are eliminated from the circulation even in the absence of antibody and that the addition of large quantities of serum from infected immunocompetent mice failed to enhance the rate or time point of removal. Any participation of complement in the process of premature removal was investigated by following the removal of ^^CrRBCs in Balb/c mice that had been decomplemented using cobra venom factor (CVF).
The use of CVF to deplete complement is a well established technique, but despite this attempts were made to confirm the decomplementation by measuring serum complement levels. Initially the CH50 method of assessing biologically active levels of complement was tested as a suitable technique; it had to be able to measure complement from normal levels down to decomplemented levels (approximately 10% or less of normal). Although the activity of mouse complement is inherently unstable this method was still tried, the blood and resultant serum being kept on ice at all times.
• — mouse semm ■— rat serum 9 0 - 8 0 - 7 0 - 6 0 - 40 - 3 0 - 2 0- t ) - T 0.01 0,001 0.1
Com plem ent dilution
Figure 5.2.a. Determination of complement activity of normal mouse (n=l ) and rat (n=3) serum by calculating CH50 values from plots of % lysis of a sensitised sRBC sample against the serum dilution.
The CH50 assay on normal mouse serum, when compared to normal rat serum, proved how inappropriate it was to use it in this situation. This method produces a relative value for the amount of complement (CH50), which is the reciprocal of the serum dilution that produces 50% lysis of a sample of sensitised sheep RBCs (sRBCs) in
comparison to total lysis (by osmotic shock) of the same volume of the sRBC sample. Values for % lysis are plotted over a range of serum dilutions, the intention being to titrate the complement activity from its plateau at maximum degree of lysis, to its plateau at minimum degree of lysis, the CH50 value can then be calculated.
The plot for the normal rat serum produced the expected sigmoidal curve, with the 100% lysis point being approached asymptotically. The CH50 value for the plot was 229.4. The plot for the normal mouse serum with the highest activity did not even pass through the 50% lysis point, even when extrapolated the y-intercept was only 70% (approximately). The highest concentration of mouse serum possible, by adding neat serum to the sensitised sRBCs, is 1:1.25 which on the extrapolated line indicated 60% lysis (approximately). Although this would be sufficient to calculate a CH50 value it would allow very little leeway; this is also a normal serum sample, a sample from a decomplemented mouse would have even lower activity. Furthermore when the activity of normal mouse serum was measured between an overall dilution of 1:1.25 to 1:16 it only gave a value of 17.8% lysis at 1:1.25, hence a CH50 value could not even be calculated. Due to the large volumes of serum that would be required for such low dilutions, and the fact that it would not be possible to obtain CH50 values for
decomplemented mice or possibly even the normal controls, this technique was deemed inappropriate.
As mouse serum is too unstable to measure complement activity, the only alternative was to measure it antigenically. A very simple and effective method was used (radial immunodiffusion (Mancini technique)), where serum diffuses through agar containing a constant concentration of anti-C3. A precipitin ring then forms with a diameter that is proportional to the concentration of C3 (complement enzyme) in the sample.
Although this technique appeared to work well, allowing levels of complement below 5% of normal to be detected, samples from the CVF treated experimental animals, taken
12 hours after the final CVF injection (d=14, 2 days after ^^CrRBC injection), showed high levels of background staining. This meant that it could not be determined whether or not the CVF treated animals had in fact been successfully decomplemented.
—o— uninfected —• — infected — infected+CW no- 9 0 - 8 0 - 70 - 6 0 - 5 0 - 40 - 3 0 - ir> 2 0- 13- 0 1 2 3 4 5
Days post infection
Figure 5.2.b. Clearance of autologous ^^Cr labelled RBCs (injected 12 days (d=12) post infection) in uninfected, infected and CVF treated infected Balb/cs. 2U of CVF injected i.v. d=12 and then 3 subsequent injections at 12 hour intervals. Values are means ± SEM (n=4).
Unfortunately due to lack of time this experiment could not be re-run. Therefore the plot of ^'CrRBC removal in CVF treated infected mice (Figure 5.2.b), which indicates no significant difference from the removal in untreated infected mice, can only be accepted as indicating a lack of complement involvement if it is assumed that the CVF did indeed cause decomplementation. If time had permitted ideally it would have been better to
develop an ELISA to detect the levels of complement, as this would have allowed very low levels to be detected antigenically. In hind-sight it would probably have been prudent to use the CH50 assays anyway, because although the complement levels could not have been quantified at least it would have given a qualitative indication of whether or not the serum concentration of complement had been reduced.