title: An enzyme-linked immunosorbant assay using monoclonal antibody against bacoside A₃ for determination of jujubogenin glycosides in Bacopa monnieri (L.) Wettst.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  The study demonstrated that ELISA using anti-bacoside A₃ MAb can be used for determination of total jujubogenin glycosides in brahmi.
authors.affiliation: Department of Pharmaceutical Chemistry and Pharmacognosy, Center of Excellence for Innovation in Chemistry, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok 65000, Thailand.
authors.forename: Charinrat
authors.initials: C
authors.lastname: Tothiam
authors.forename: Watoo
authors.initials: W
authors.lastname: Phrompittayarat
authors.forename: Waraporn
authors.initials: W
authors.lastname: Putalun
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Tanaka
authors.forename: Seiichi
authors.initials: S
authors.lastname: Sakamoto
authors.forename: Ikhlas A
authors.initials: IA
authors.lastname: Khan
authors.forename: Kornkanok
authors.initials: K
authors.lastname: Ingkaninan
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D034681:Bacopa / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D035261:Plant Components, Aerial / Q000737:chemistry; D015203:Reproducibility of Results; D012503:Saponins / Q000032:analysis* / Q000276:immunology; D012680:Sensitivity and Specificity; D014315:Triterpenes / Q000032:analysis* / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D012503:Saponins; D014315:Triterpenes; C087625:bacoside A3