Immunoassay – a green chemistry for phytochemical analysis

Walailak Frontier 04, October 2018


Immunoassay – a green chemistry for phytochemical analysis


Immunoassay for herbal standardization


Immunoassays are an analytical method based on principle of a specific interaction between an antibody and its antigen. The assays are broadly applied for medical diagnosis and researches. Now, the immunoassays were developed for analyses of phytochemicals, the chemical constituents produced by plants. A quantity of phytochemicals consumed directly affects the efficacy and safety of herbal products, because these compounds mediate pharmacological effects. Analyses of a phytochemical content using conventional chromatographic techniques are limited by their low sensitivity and specificity. In addition, the techniques require complex instruments and a large quantity of organic solvents which are of personal safety and environmental concerns. 

Kwao Krua Kao or Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham is a high potential herb for menopause remedy. For optimal efficacy and safety, the daily consumed amount is restricted according to its potent estrogenic contents of miroestrol and deoxymiroestrol. Therefore, analyses of both compounds are required. Determination using HPLC-UV of both compounds in plant extract matrix is quite difficult due to chemical interferences, especially when Kwao Krua Kao was prepared in a mixed formulation with various herbs. To simplify analytical method of miroestrol and deoxymiroestrol, a specific monoclonal antibody against miroestrol and deoxymiroestrol is developed using hybridoma technology. The antibody interacts specifically against miroestrol and deoxymiroestrol, but its cross-reactivities forward to other compounds of Kwao Krua Kao are very low. The sensitivity of determination by an enzyme-linked immunosorbent assay (ELISA) protocol is in the range of 31.3 – 500 ng/mL (50 times higher sensitivity than HPLC-UV) with high precision. Besides the highly sensitive and specific binding manners, analytical protocol of the ELISA does not require sample pre-treatment steps and large quantity of toxic organic solvents. The ELISA is applicable for Kwao Krua Kao either in raw materials, intermediate-extracts, or finish goods. Therefore, we can ensure quality of Kwao Krua Kao derived products. In addition, immunochromatographic strip test is another example of simple and effective immunoassay format for herb identification. Totally, immunoassay is green chemical approach with high performance for herbal standardization.


  • Yusakul, G., Kitisripanya, T., Juengwatanatrakul, T., Sakamoto, S., Tanaka, H., Putalun, W., 2018. Enzyme linked immunosorbent assay for total potent estrogenic miroestrol and deoxymiroestrol of Pueraria candollei, a Thai herb for menopause remedy. J Nat Med 72, 641 – 650

  • Yusakul, G., Putalun, W., Udomsin, O., Juengwatanatrakul, T., Chaichantipyuth, C., 2011. Comparative analysis of the chemical constituents of two varieties of Pueraria candolleiFitoterapia 82, 203-207

  • Rose, A.S., Bradley, A.R., Valasatava, Y., Duarte, J.M., Prlić, A., Rose, P.W., 2018. NGL viewer: web-based molecular graphics for large complexes, Bioinformatics, pp.419-419

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