Complement Heat Tolerance as a Marker of Protein Fragility and Its Clinical Significance

Abstract

Objective: This study aimed to establish a complement-tolerance test as a marker of protein fragility and discuss its clinical significance. Methods: Total complement activity (TCA) of serum was measured using a self-hemolysis colorimetric method. Human O-erythrocytes and rabbit anti-human O-erythrocyte antibodies were used to replace sheep erythrocytes and the corresponding hemolysin for the hemolysis test, respectively. The antigen-antibody specific binding activated the classical pathway of complement, generating a membrane attack complex (MAC), and the red blood cells rupture. A complement-tolerance test (CTT) was established to measure complement heat tolerance according to the sensitivity of complement proteins to temperature, which was calculated according to differences in TCA at different temperatures. The smaller the CTT, the stronger the complement resistance to heat. The method was applied to the detection of diabetic patients and healthy controls. Results: The mean value of CTT (Mean) = 0.063 ± 0.003 with a coefficient of variation of 4.8% for the same specimen tested for complementary thermal resistance on five consecutive days, which is a good stability of the assy. Application of CTT on samples from patients with different ages revealed significantly higher mean CTT values for elderly patients (≥60-years old) relative to those for younger patients (20–40-years old) (p < 0.05). Additionally, the mean CTT values for diabetic patients were significantly higher than those for healthy patients (p < 0.001). Conclusion: We successfully established a method that uses complement thermal resistance as a marker of protein fragility, with the results demonstrating the ability of the CTT identify age- and disease-related variations in patient samples and its potential efficacy for clinical application.