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1School of Pharmacy and Medical Sciences, University of South Australia
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The classical pathway is activated by antibody and culminates in target cell lysis. The CH50 assay provides a measure of the complement activity of a serum sample. This video demonstrates the steps involved in determining the CH50 of a serum sample, the calculations and interpreting of results.
Costabile, M. Measuring the 50% Haemolytic Complement (CH50) Activity of Serum. J. Vis. Exp. (37), e1923, doi:10.3791/1923 (2010).
Preparation of 5x Veronal Buffered Saline (VBS)
Sensitisation of sheep red blood cells with haemolysin
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.
|Total lysis||0.183, 0.183||0.183|
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.
The CH50 assay is subject to many interferences. Some SRBC are more fragile than others, resulting in spontaneous haemolysis that is unrelated to complement activity. The affinity of the rabbit antibody varies from lot to lot and from one manufacturer to another; this affects the amount of antibody that binds to the SRBC. Also, the process of sensitising SRBC with antibody results in cells with differing amounts of antibody coating the SRBC. Specimen collection and storage are an important potential source of error. Complement components e.g. C1q, C3, C4 and C5 are extremely labile, so proper sample handling is critical. Prolonged exposure to heat will decrease complement activity and will produce inactive fragments of complement components. To detect as many sources of error as possible, it is critical to test a control serum with a known CH50 value every time the assay is performed and to reproduce the accepted value of the known serum control. One way to determine if there are any differences between different batches of SRBC and haemolysin is to test new test materials against a standard sample of serum several times and then determine if there are any changes in the basal level of haemolysis or in the CH50 value for the control serum.
The author would like to thank Miss Lora Matthews for performing the technique and Mr Paul Shepherd for cinematography. This project was funded by the University of South Australia, Laboratory Medicine program.
|Sheep red blood cells||CSL||2490201||Use at 1% final|
|Rabbit Anti-Sheep Haemolytic serum (RBCs), Unconjugated||AbD Serotec||C12HSB|
|96 well flat bottom plate||Sarstedt Ltd||83.1839|
|Veronal buffered saline||Use at 1x final|
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