[Preparation of monoclonal antibodies against saikosaponin A and saikosaponin D and establishment of indirect competitive enzyme-linked immunosorbent assay].

Saikosaponin A(SSA) and saikosaponin D(SSD) were conjugated with bovine serum albumin(BSA) and ovalbumin(OVA) via the periodate oxidation method to prepare immunogens(SSA/D-BSA) and coating antigens(SSA/D-OVA), respectively. BALB/c mice were immunized with SSA/D-BSA, and 15 monoclonal antibody(mAb) cell lines against SSA/D were screened. Among these, the mAb SD6 against SSD exhibited cross-reaction rates of 100.00%, 99.94%, 95.06%, 94.89%, 1.97%, 1.89%, and 0.93% with SSD, SSA, 6″-O-acetyl-saikosaponin A, 6″-O-acetyl-saikosaponin D, saikosaponin C(SSC), saikosaponin E(SSE), and hydroxysaikosaponin A, respectively. The cross-reactivity rates with saikosaponin F(SSF), saikosaponin B1(SSB1), saikosaponin B2(SSB2), and other compounds were all less than 0.10%. An indirect competitive enzyme-linked immunosorbent assay(icELISA) based on the selected mAb SD6 against SSD demonstrated an IC_(50) value of 32.68 ng·mL~(-1), a detection range from 6.4 to 159.12 ng·mL~(-1), a spiked recovery from 95.03% to 104.5%, and RSD from 2.7% to 4.0%. The developed icELISA was applied to determine the total content of SSA and SSD in 19 batches of Bupleurum chinense DC. The results were validated using ultra-high-performance liquid chromatography(UPLC), with a high correlation coefficient(0.994 7) shown between the two methods. This confirms that the established icELISA is reliable for rapid quantification of the total SSA and SSD content in B. chinense.