Inter-Individual Variations: A Challenge for the Standardisation of Complement Activation Assays

Figures & data

Figure 1 Overview of the whole workflow in the experimental setup: Selection of complement activators; Quality assessment; Complement activation.

Figure 2 Serology testing protocol for evaluation of potential complement activation effects of test samples (ie, liposomes) in human serum.

Figure 3 iC3b levels measured in pooled human serum incubated with positive and negative controls.

Figure 4 Variability of iC3b complement fragment determined in healthy donors compared to pooled serum.

Notes: The dot plot demonstrates individual responses after activating the human iC3b complement fragment in sera of 32 healthy donors. The sera were incubated with 10 units/mL CV for 1h and compared with commercially available pooled serum. The horizontal grey bar represents the range of the average amount of iC3b cleavage in pooled serum. The mean value of 32 responses (pink cross) shows that this value is still in range of pooled serum, which was experimentally obtained.

Figure 5 iC3b levels in sera of donors with allergy, immunodeficiency or cancer disease exposed to cobra venom.

Notes: Complement activation in serum of donors with allergy, immunodeficiency or cancer disease exposed to cobra venom grouped by blood type (A) and by gender (B). In (A) the open symbols correspond to donors with different allergies, the solid symbols to donors with immunodeficiency and the cyan star is the mean value of the 15 donors suffering from breast or lung cancer, of which we do not know the blood type. The pink symbols represent the average of all donors with allergy (open star) and all donors with immunodeficiency (cross). The horizontal lines correspond to the activation of pooled serum from healthy donors. In (B) the cyan stars correspond to the iC3b activation of serum from each individual donor with cancer.

Figure 6 Liposome inducing changes in complement activation product iC3b measured in sera of healthy donors.

Notes: (A) Dose response curves of activation of the complement system triggered by three different liposomes in pooled serum of healthy donors. (B) Complement activation by two concentrations of the liposome with the highest potential to induce an adverse immune response evaluated in sera from individual healthy donors. Data are grouped by blood type. The average of iC3b released upon exposure to 50 and 150 µg/mL of F20104A liposome is represented by the open pink star and the cross, respectively. The horizontal lines correspond to the iC3b levels observed in pooled sera from healthy donors after exposure to 50 µg/mL (bottom line) and 150 µg/mL (top line) of liposomes. The grey band represents the responses in pooled serum.

Figure 7 Liposome induced iC3b cleavage after treatment of sera from donors with pre-existing conditions.

Notes: Liposome F20104 increasing the iC3b amount after treatment of sera from donors with preconditions, grouped by blood type for donors with allergy (open symbols) and immunodeficiency (solid symbols). The cyan star is the mean value of the 15 donors suffering from breast or lung cancer. The pink symbols represent the average of all donors with allergy (open star) and all donors with immunodeficiency (cross). The horizontal lines correspond to the activation of pooled serum from healthy donors.