Preparation of 5x Veronal Buffered Saline (VBS)
Sensitisation of sheep red blood cells with haemolysin
CH50 assay
Calculations
Representative Results:
The video includes an example of representative results. In practice, a control serum is also run at the same time as the test serum and is treated in the same manner. Below (Table 1) is sample data from a tested serum sample. The data is manipulated according to the equation presented in 5.3.
Sample | OD540 | Mean | ||
Blank | 0.042, 0.044 | 0.043 | ||
Total lysis | 0.183, 0.183 | 0.183 | ||
Dilution | OD540 | Mean | Mean-Blank | % Lysis |
1:8 | 0.200, 0.219 | 0.210 | 0.168 | 116 |
1:16 | 0.179, 0.173 | 0.176 | 0.134 | 93 |
1:32 | 0.134, 0.110 | 0.122 | 0.08 | 55 |
1:64 | 0.053, 0.066 | 0.059 | 0.017 | 12 |
1:128 | 0.044, 0.045 | 0.045 | 0.003 | 2 |
Figure 1. Activation of the classical complement pathway. The classical pathway is activated by binding immunoglobulin-M (IgM) or immunoglobulin-g (IgG) on the surface of a target cell. The Fc portion of the Ab binds to C1q, C1r is activated and this in turn activates another molecule of C1r which together activate two molecules of C1s. C1s now cleaves C4 which exposes the binding site for C2 which is also cleaved. The binding of C4b and C2a leads to the formation of a complex referred to as a C3 convertase. This complex now cleaves C3 forming C3a and C3b, some of which combine with the C3 convertase forming a C5 convertase. This complex now acts on C5, with the resulting C5b binding to C6 initiating the formation of the membrane attack complex (MAC). C5b6 acts on C7, which in turn act on C8 and ultimately on C9 resulting in the formation of the final MAC. (Goldsby et al, 2003).
Figure 2. Plotting of sample data and calculating the CH50 for a control and test serum sample. (A), The calculated percentage (%) lysis of both the control (•) and test () serum samples are plotted against dilution factor. (B) To calculate the 50% lysis, a line is drawn from the 50% percent value until it intersects with the graph lines and then a vertical line is drawn down to the dilution. In this example, a 35-fold dilution of the control and 21.6-fold dilution of the test serum are required to achieve 50% lysis. This data indicates that there is less complement present in the test serum compared to the control as it required less dilution before 50% lysis was reached. The control sample also shows > 100% lysis at the 1:8 dilution. This is most likely due to incomplete lysis of the SRBC by the Distilled water and more efficient lysis by the complement components.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Sheep red blood cells | CSL | 2490201 | Use at 1% final | |
Serum | Human serum | |||
Rabbit Anti-Sheep Haemolytic serum (RBCs), Unconjugated | AdB Serotec | C12HSB | ||
96 well flat bottom plate | Sarstedt | 83.1839 | ||
Veronal buffered saline | Use at 1x final | |||
Waterbath | ||||
Plate reader | ||||
37°C room/incubator |