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

Charinrat Tothiam, Watoo Phrompittayarat, Waraporn Putalun, Hiroyuki Tanaka, Seiichi Sakamoto, Ikhlas A. Khan, Kornkanok Ingkaninan

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)385-391
Number of pages7
JournalPhytochemical Analysis
Volume22
Issue number5
DOIs
Publication statusPublished - Sep 1 2011

Fingerprint

Bacopa
Bacopa monnieri
Glycosides
glycosides
Assays
monoclonal antibodies
Monoclonal Antibodies
assays
Enzymes
enzymes
Saponins
bovine serum albumin
Bovine Serum Albumin
saponins
cross reaction
Ayurvedic Medicine
Ayurvedic medicine
Immunization
haptens
myeloma

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Food Science
  • Biochemistry
  • Molecular Medicine
  • Plant Science
  • Drug Discovery
  • Complementary and alternative medicine

Cite this

An enzyme-linked immunosorbant assay using monoclonal antibody against bacoside a3 for determination of jujubogenin glycosides in Bacopa Monnieri (L.) wettst. / Tothiam, Charinrat; Phrompittayarat, Watoo; Putalun, Waraporn; Tanaka, Hiroyuki; Sakamoto, Seiichi; Khan, Ikhlas A.; Ingkaninan, Kornkanok.

In: Phytochemical Analysis, Vol. 22, No. 5, 01.09.2011, p. 385-391.

Research output: Contribution to journalArticle

Tothiam, Charinrat ; Phrompittayarat, Watoo ; Putalun, Waraporn ; Tanaka, Hiroyuki ; Sakamoto, Seiichi ; Khan, Ikhlas A. ; Ingkaninan, Kornkanok. / An enzyme-linked immunosorbant assay using monoclonal antibody against bacoside a3 for determination of jujubogenin glycosides in Bacopa Monnieri (L.) wettst. In: Phytochemical Analysis. 2011 ; Vol. 22, No. 5. pp. 385-391.
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abstract = "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.",
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AU - Putalun, Waraporn

AU - Tanaka, Hiroyuki

AU - Sakamoto, Seiichi

AU - Khan, Ikhlas A.

AU - Ingkaninan, Kornkanok

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N2 - 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.

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