Hemolytic Assay Protocol for C2

Introduction of C2 and Hemolytic Assay

In humans, complement component 2 (C2), a serum glycoprotein, is encoded by the C2 gene located on the short arm of chromosome 6. C2 functions as a multi-domain serine protease in the classical pathway of the complement system and plays a critical role in the immune response. After the cleavage by activated C1, C2 will separate into C2b and C2a, the latter will bind to the activated surface-bound C4b to create the C3 or C5 convertase in a Mg²⁺-dependent way. The hemolytic assay can be used to evaluate the strength and toxicity of different chemicals such as C2 and to quantify this capacity by the chemical-inducing lysis of red blood cells.

Materials for Hemolytic Assay

• Normal human serum (NHS)
• Normal human C2-depleted serum (C2D)
• HEPES-buffered saline buffer
• 2% Antibody-Sensitized Sheep Erythrocytes (SRBCs)
• 96-well U-bottomed plate

Protocol of Hemolytic Assay for C2

1. In a 96-well U-bottom plate, prepare a serial dilution of NHS.
2. Repeat step a for serial dilution of C2D.
3. Prepare the 0% lysis controls (HBS) and 100% lysis controls (ddH₂O).
4. Add HBS in each well except the well containing ddH₂O. Add ddH₂O to this well.
5. Add 2% SRBCs to each well.
6. Seal and incubate the plate at 37°C.
7. Centrifuge the plate to stop the reaction.
8. Transfer the supernatant of each well to a new plate without disturbing the pelleted SRBCs and determine the absorbances at 405 nm by a plate reader.
9. Verify controls to show 0% (Background) and 100% (Max) lysis, respectively.
10. Calculate the % Lysis in samples and measure the 50% hemolytic complement (CH50) activity.

Fig.1 Hemolytic assay protocol for C2.

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