Complement-Mediated Hemolysis Protocol

Complement-Mediated Hemolysis (TNP-Specific)

Sheep red blood cells with covalently attached TNP to their cell surface, were utilized in this complement-mediated lysis to verify which B cell hybridomas produced functional IgM capable of initiating the complement cascade leading to the deposition of the membrane attack complex (MAC) on the surface on the red blood cells, leading to hemolysis, when all complement components are present (at adequate amounts). Any supernatant IgM that could mediate complement mediated hemolysis would require the IgM to have an unaltered, non-mutated (mutations that would affect its specificity)) V-region be capable of binding to their specific antigens, TNP. In addition, C domains, functionable, binds the C1q complement component, and is therefore responsible for IgM ability to activate MAC and cause lysis. Sp6 supernatant was only capable of causing lysis of the TNP conjugated cells. The lysis was seen as a visible transparent clearing in the uniform sheep red blood cell flat surface, which was a circle that was seen as more transparent, a clearing of 'ghost' cells, than compared to the surroundings. Moreover, a gradient in the size of the clearing was seen. The diameter of the 'ghost' clearings increased in size, from 5 mm to 7 mm, as the concentration of supernatant increased.

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