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

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.

Hemolytic assay protocol for C2. (Creative Biolabs Original) Fig.1 Hemolytic assay protocol for C2.

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Published Data

rhC2 characterization.Fig 2. Characterization of recombinant human complement C2. 1

C2 plays an important role in innate immunity and immune tolerance. Complement C2 deficiency is associated with a variety of diseases, the most prominent of which are recurrent severe infections in young children and systemic lupus erythematosus (SLE) in adults. Recombinant human complement C2 (rhC2) has been cloned and expressed in mammalian cell lines with a purity of more than 95% and characterized for stability and activity. Recombinant human C2 is sensitive to cleavage by C1s and can restore the activity of the classical complement pathway in C2-deficient sera in complement activation ELISA and hemolysis assays. In addition, rhC2 was found to increase the deposition of the C3 fragment of the human pathogen Streptococcus pneumoniae in C2-deficient sera to the same level as in normal sera. These results suggest that recombinant human C2 may be a potential therapy for recurrent bacterial infections or systemic lupus erythematosus in patients with C2 deficiency.

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Reference

  1. Martini, Paolo GV, et al. "Recombinant human complement component C2 produced in a human cell line restores the classical complement pathway activity in-vitro: an alternative treatment for C2 deficiency diseases." BMC Immunology 11 (2010): 1-10. Distributed under Open Access license CC BY 2.0, without modification.
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