Development of a Complement Hemolysis Assay Using Aldehyde-Modified Human Erythrocytes

BackgroundComplement-mediated hemolysis assays are essential for assessing immune function and diagnosing complement-related disorders. Conventional human erythrocyte derivatization with 2,4,6-trinitrobenzene sulfonic acid (TNBS) can induce nonspecific hemolysis and optical interference, complicating interpretation. Identifying a more biocompatible electrophile could improve assay specificity and reliability. MethodsA panel of aldehydes was screened for electrophilicity using a nucleophile consumption assay with glycine as a model nucleophile. Glyoxylic acid was selected based on reactivity, solubility, and visual neutrality, then neutralized with sodium bicarbonate to minimize baseline hemolysis. Human erythrocytes were sequentially treated with pancreatin and glyoxylic acid to generate glyoxylic acid-pancreatin-treated erythrocytes (GxPEs). Complement-mediated hemolysis was assessed using normal human serum, heat-inactivated serum, and pathway-specific conditions, with CH50 values calculated for total, alternative, and non-alternative pathways. ResultsGxPEs exhibited robust complement-specific hemolysis (maximum 93.56%) with negligible background activity in heat-inactivated serum. CH50 analysis confirmed activation via both alternative (0.9514 L) and non-alternative (1.963 L) pathways. Reconstitution experiments with factor B-depleted cryoprecipitate and cryosupernatant fractions demonstrated dependence on small complement components such as C2 and C4. ConclusionsGlyoxylic acid derivatization yields a reproducible, optically quiet, and complement-specific erythrocyte substrate suitable for functional hemolysis assays. This method offers a practical platform for complement diagnostics, research applications, and therapeutic evaluation.