Complement Mediated Hemolysis
The complement mediated hemolysis test is based on the ability of serum antibodies to form complement-activating immune complexes upon union with their specific antigens. Within immune complexes, C1q complement component, binds to neighbouring Fc regions of antibody molecules with at least two of its globular head domains. This primary interaction produces a cascade of proteolytic cleavage reactions, which consume complement components. However, these reactions eventually lead to the deposition and formation of a membrane attack complex, MAC, which eventually forms pores within the target cell and destroys the cell, through the destruction of any gradients and membranes, leading to cell death. However, the test sera must have its complement heat-inactivated and that the terminal components of complement have led to the MAC (1,2,3).
Within our experiment, sheep red blood cells were covalently attached with TNP to their cell surface, as lysine residues and primary and secondary amines. These TNP-SRC were further mixed with guinea pig complement and mixed with liquid agarose, forming an indication layer system upon solidification. This mixture layer, plated and solidified, was used to identify any TNP-specific antibody capable of activating complement, through visible lysis of the SRC-TNP, caused by the formation of the membrance attack complex, at the point of supernatant application. Any lysis, seen as a circle of "ghost" cells, would indicate that the serum contained TNP specific IgM, capable of activating complement through having a functional Fc region. More specifically, the IgM would have to have variable regions (V-regions) capable of binding to TNP and a CH3 domain (Fc portion) able to activate complement, by binding C1q and activating it to initiate complement (IgM needs to be in a polymeric form in order to activate complement. CH3 is not intrinsically active in C1q binding, but requires strain from going into the staple formation that relieves masking effect of CH4 on CH3. Distortional force is transmitted through CH2 from Fabs upon binding antigen, in staple form, complete C1q binding site in CH3. CH2 is known to act as a domain of bonding, as in classic allosteric effects (5).
The complement-mediated lysis test fails if the supernatant consumes complement without antigen addition, due to the presence of pre-existing complement-activating factors in the sera, such as immune complexes. Moreover, in mutants, such as polyclonal B cell activation in patients with Epstein-Barr virus infection, the B cells may generate antibodies against sheep red blood cells that can give false positive results (3).
1. Janeway C.A., Travers P., Walport M., and Capra J.D. 1999. Immunobiology: The immune system in health and disease. Garland Publishing, 4th ed., New York, USA pages 1-40, 2.5-2.22, 3.1-3.12
2. Delves P., and Roitt I. 1999. Encyclopedia of Immunology. Academic Press Inc., 2nd ed., San Diego, USA
3. 1994 Current Protocols in Molecular Biology. Volume 2. John Wiley & Sons Inc., USA
4. Cruse J., and Lewis R. 1995. Illustrated Dictionary of Immunology. CRC Press Inc., USA pages 1960-1965
5. Chen F.H., and Painter R.H. 1997. Domain switched mouse IgM. IgG2b hybrids indicate individual roles for C domains in the regulation of the interactions of IgM complement C1q. J. Immunol. 159, 3354-3363
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