Production, characterization of a monoclonal antibody against aristolochic acid-II and development of its assay system

Aristolochic acid-II (AA-II) conjugated with bovine serum albumin (BSA) was used as an antigen for immunizing BALB/c mice. Isolated splenocytes from the immunized mice were fused with an aminopterin-sensitive mouse myeloma cell line, SP2/0-Ag14, to produce hybridoma cells that secreted a monoclonal antibody (MAb) against AA-II. The selected hybridoma was subsequently cloned by limited dilution method. For MAb, the isotype and an estimated dissociation constant (KD) of the MAb were determined. The MAb was used to establish an ELISA method. Accuracy and variation assays, as well as determinations of the specificity and sensitivity, were also carried out and the linear range was 0.19-13 mu g/ml. The anti-AA- II MAb showed a very high specificity for AA-II and had low cross-reactivities against the other aristolochic acid (AAs) (CR: AA-I, 3.4%; AA-VIIa, 0.86%) or aristololactam-I (AL-I) (CR < 0.07%) except AA-IIIa which has 17% of cross activity. Anti-AA-II MAb also showed negligible cross-reactivity (< 0.5%) toward other natural compounds with different chemical structures including barbaloin, sennoside A, rutin, glycyrrhizin, caffeic acid etc. This is the first time that an ELISA method was successfully established for the application of anti-AA-II MAb.