title: Development of a C3c-based ELISA method for the determination of anti-complementary potency of Bupleurum polysaccharides.
abstract: Traditionally, determination of inhibitory potency of complement inhibitors is performed by the hemolytic assay. However, this assay is not applicable to the lectin pathway, thus impeding the understanding of complement inhibitors against the overall function of the complement system. The main objective of our study was to develop a specific enzyme-linked immunosorbent assay (ELISA) as an alternative method to assess the anti-complement activity, particularly against the lectin pathway. By using respective coating substrates against different activation pathways, followed by capturing the stable C3c fragments, our ELISA method can be used to screen complement inhibitors against the classical pathway and the lectin pathway. The inhibitory effect of suramin on the classical pathway, as measured by our hemolytic assay is consistent with previous reports. Further assessment of suramin and Bupleurum polysaccharides against the lectin pathway showed a good reproducibility of the method. Comparison of the lectin pathway IC50 between Bupleurum smithii var. parvifolium polysaccharides (1.055 mg/mL) and Bupleurum chinense polysaccharides (0.98 mg/mL) showed that, similar to the classical and alterative pathway, these two Bupleurum polysaccharides had comparable anti-complementary properties against the lectin pathway. The results demonstrate that the described ELISA assay can compensate for the shortcomings of the hemolytic assay in lectin pathway.
authors.affiliation: Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, China.
authors.forename: Mulu
authors.initials: M
authors.lastname: Wu
authors.affiliation: Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, China.
authors.forename: Yunyi
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 201203, China.
authors.forename: Daofeng
authors.initials: D
authors.lastname: Chen
keywords: AP, alternative pathway; Abs, antibodies; BCPs, Bupleurum chinense polysaccharides; BG, background value; BPs, Bupleurum smithii var. parvifolium polysaccharides; BSA, bovine serum albumin; Bupleurum chinense; Bupleurum smithii var.  parvifolium; CP, classical pathway; CV, coefficient of variation; Complement C3c; DFC, drug-free control; ELISA; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxides; LP, lectin pathway; LPS, lipopolysaccharide; MASP, MBL-associated serine proteases; MBL, mannose-binding lectin; OD, optical density; PBS-T-BSA, PBS containing 0.05% Tween-20 and 1% BSA; Polysaccharides; SRBC, sheep erythrocytes; Suramin; VBS, Veronal buffer saline