An enzyme-linked immunosorbant assay using monoclonal antibody against bacoside a3 for determination of jujubogenin glycosides in Bacopa Monnieri (L.) wettst

Introduction - In Ayurvedic medicines, Bacopa monnieri (L.) Wettst. (brahmi) is known as a medicinal plant used for memory enhancement. Its active compounds are classified as pseudojujubogenin and jujubogenin glycosides. Owing to the lack of chromophore in the saponin glycoside structures, HPLC-UV-vis gives low sensitivity for determination of such compounds. In the case of the detection of small amounts of saponin glycosides, immunological assay could be a suitable method. Objective - To develop and validate a sensitive enzyme-linked immunosorbant assay (ELISA) using monoclonal antibody (MAb) against bacoside A3, the major jujubogenin glycoside found in brahmi. Methodology - An immunogen was prepared by conjugating bacoside A3 with a bovine serum albumin (BSA). To determine its immunogenicity, the ratio of hapten in bacoside A3- BSA conjugate was determined by matrix-assisted laser desorption/ ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). After immunisation in mice, hybridomas secreting MAbs against bacoside A3 were produced by fusing the immunised splenocytes with SP2/0- Ag14 myeloma cells. The antibody was raised specifically against jujubogenin glycosides. The ELISA using anti-bacoside A3 MAb was developed. Results - Bacoside A3 in the range of 3.05-97.70 ng mL-1 could be detected by ELISA using anti-bacoside A3 MAb. The assay showed a detection limit of 0.48 ng mL-1 (0.517 nM). The validation study showed that the method was precise, accurate and sensitive. Interestingly, the MAb showed cross-reactivity with the other jujubogenin glycosides, bacopaside X and IV. However, it did not show cross-reactivity with any of pseudojujubogenin glycosides. Conclusion - The study demonstrated that ELISA using anti-bacoside A3 MAb can be used for determination of total jujubogenin glycosides in brahmi.