Serum haemolytic activity, C3 levels and electrophoretic changes in C3 levels in normal human sera stored at 4 degrees C.
C3 is a pivotal substance in the complement system. Low values of serum C3 are interpreted as an indication of utilization of C3 in the complement cascade. Mere storage of normal serum is well known for the concomitant reduction of haemolytic activity, but the C3 levels do not change. Immunoelectrophoretic analyses have shown a conversion of C3 on addition of antigen-antibody complexes from a B1C to B1A (electrophoretically faster moving) form. This change can be elegantly demonstrated by use of two dimensional antigen-antibody electrophoresis (2-DCIEP). The change also occurred when sera were treated with hydrazine.,  These observations suggest that utilization or activation of C3 may only lead to a change in its electrophoretic properties. Total serum C3 levels were not measured in any of these studies. We describe here an attempt at correlating the haemolytic activity of serum, the C3 content and the electrophoretically distinct forms of C3 in normal human sera and changes with storage at 4°C.
These were obtained from 10 normal healthy adults. They included five males and five females. The mean age was 26.7 years (range 24 to 38 years). Ten ml of blood were collected aseptically in a sterile test tube and a small aliquot of separated serum was removed 30 minutes after collection. This was studied with and without inactivation (at 56°C for 30 minutes) by the methods described below. Similarly, the remaining serum was collected, stored at 4°C and studied at 24 hours, 3 days, 7 days, 13 days and on the 25th day.
This was determined as described by Kotwal and Kelkar. Neat serum and doubling dilutions (1 in 2, 4, 8, 16 and 32) in volumes of 0.1 ml were dispensed in plastic microtiter plates. To each was added 0.1 ml of a two per cent suspension of sheep red cells sensitized with two minimum haemolytic doses of amboceptor (Haffkine Biopharmaceuticals, Bombay). The plates were incubated at 37°C for 30 minutes and at 4°C for four hours and readings taken. The end point was the highest dilution that showed complete lysis of the red cells. Results were expressed in a linear fashion with each dilution being given a unit value. Thus an end point titre of 1 in 2 rated 2, 1 in rated 3, 1 in 8 rated 4, 1 in 16 rated 5 and 1 in 32 rated 6 units.
This was tested as described by Kotwal and Kelkar.
Quantitation of C3 by radial immunodiffusion (RID)
This was done as described by Mancini et al. Monospecific antibody was obtained from Immunodiagnostic Laboratories, New Delhi. The standard was locally calibrated from a master standard obtained from Hyland Laboratories.
Two dimensional antigen antibody electrophoresis (2-DCIEP)
This was carried out on a glass plate 85 x 75 mm. as described earlier. All ten samples were studied simultaneously on one plate. Monospecific anti-C3 was incorporated in a concentration of 1 in 30 in the second dimension. The two components B1C and B1A formed distinct peaks. The relative proportions of each were quantitated by tracing photographic enlargements of the separations on paper, cutting them and weighing the two pieces.
None of the sera at any stage showed anticomplementary activity. [Table - 1] summarises the results. [Fig. 1] (see page ... ) is a scatter diagram giving the detailed values obtained. Fresh sera showed a mean haemolytic activity of 3.3 ± 2.3 units (range 0 to 5 units). The C3 level was 184.8 ± 44 mg/dL. 2-DCIEP showed all sera to contain only the BIC component. Inactivated sera showed complete loss of haemolytic activity; the mean C3 level was 144.4 ± 37 mg/dl, the difference being statistically significant (p< 0.05). 2-DCIEP showed all the sera to contain only the BIA component. Sera stored at 4°C showed a gradual loss of haemolytic activity. There was a significant fall by day 13 and by day 25 the haemolytic activity had almost disappeared. Serum C3 levels, however, did not show any significant changes. 2-DCIEP showed a gradual conversion of B1C to B1A form (see [Fig. 2] on page ...).
[Table - 1]
Linscott and Cochrane studied C3 in guineapig sera by use of immunoelectrophoresis and by determining the haemolytic activity of the sample. In the guineapig, mouse and rat, C3 is electrophoretically faster-moving but becomes slow when inactivated. Addition of immune complexes to the serum sample led to both conversion of C3 to the slower-moving form and to a loss of haemolytic activity. Similarly, mere storage of serum for four to six weeks at 4°C led to a gradual progressive and finally complete conversion of C3 to its inactive form and to a loss of haemolytic activity.
Laurell and Lundh investigated human sera to determine the conversion of C3 from the BIC to BMA forms. They used 2-DCIEP for this demonstration. Sera were studied sequentially after storage for four days, after addition of immune complexes, hydrazine and ethylene diamine tetra acetic acid (EDTA) and after heating at 56°C for half an hour. Fresh sera showed only the BIC form of C3. Storage at 37°C for four days caused a complete conversion of C3 to the BIA form. Similarly heating at 56°C or treating with immune complexes or hydrazine led to a similar conversion. EDTA did not stabilize C3.
The present study has brought out quite clearly that mere determination of C3 by radial immunodiffusion does not reflect its haemolytic activity. Storage of normal sera for 25 days did not cause any quantitative changes in C3 levels but was associated with a progressive conversion of C3 from B1C to B1A form. In view of the concomitant change in haemolytic activity, it is reasonable to interprete the B1C form as active and B1A form as inactive C3. Of course, other complement components may be critical in the loss of haemolytic activity. Sera heated at 56°C for half an hour showed levels of C3 significantly lower than the levels in fresh sera. This might be on account of the denaturation of some of the C3 because of heat. C3 exists in immunologically similar but electrophoretically and functionally different forms. This is an important observation because the conventional view of consumption of complement visualises the reduction or the disappearance of C3. In reality it is not a consumption but a conversion of an active to an inactive form. A corollary of this observation would mean that mere determination of C3 levels in a sample of serum by an immunological method would be of little utility in measuring the consumption of C3 in immunological reactions. Preliminary observations in sera of cases of typhoid fevers have shown a considerable conversion of C3 from the B1C to B1A form in fresh sera.