| Objective: Flos Lonicerae Japonicae(FLJ, Jin-Yin-Hua in Chinese), the flower buds of Lonicera japonica Thunb.(Caprifoliaceae), is a widely used traditional Chinese medicine with various pharmacological activities. Currently, it has been widely added in beverages, health products, food and cosmetics owing to its proven health benefits and low toxicity. However, the quality of FLJ varies considerably with species, growing regions, climatic conditions, cultivars, harvest time and processing. Therefore, a reliable, simple, rapid and high-throughput method is required to screening the authentication and quality of FLJ on the market in order to guarantee the stable therapeutic effect and safety. The Enzyme-linked immunosorbent assay(ELISA), characterized with simple, rapid, reliable, low costs, and high throughout, may supply an alternative analytical procedure for the detection of the quality of FLJ.Methods: Chlorogenic acid(CGA) and luteoloside are the two major active compounds and quality control markers of FLJ. In this paper, two specific monoclonal antibodies against CGA(2E2) and luteoloside(3A4) were produced by hybridoma technology, respectively. ELISA methods were developed based on the artificial antigens and monoclonal antibodies. Rapid and sensitive colloidal gold immunochromatography test strips(CGIC) based on the monoclonal antibodies were also developed.Results: The complete antigen of CGA and luteoloside were synsthsised by active ester method and confirmed by UV method. A monoclonal antibody-based effective ic ELISA was developed and applied for the detection of CGA, IC50 of which was 0.4 ng/m L, calibration range was 0.10-1.51 ng/m L, and there was relative high cross-reactivity(CR) only with 3,5-Dicaffeoylquinic acid(17.53%), and CRs with other analogies were all below 5% and regarded as negligible. The recoveries obtained by standard CGA addition to FLJ samples were from 88.4% to 104.8%. An ic ELISA was also established based on the monoclonal antibody against luteoloside. The concentration of luteoloside producing 50% inhibition and the working range of ic ELISA were 42.3 μg L-1 and 9.1-258.1 μg L-1, respectively. The ic ELISA showed cross-reactivity(CR) of 2414%, 402%, 230% and less than 1% with luteolin-7-O-glucuronide, baicalin, scutellarin and other analogs of luteoloside. The average recoveries of luteoloside in FLJ samples determined by ic ELISA ranged from 82.9% to 112.5%.The CGIC dipstick has been used in estimation of the quality of FLJ samples. The dipstick for CGA was developed with the detection limit of 50~100 ng/m L. The dipstick showed a relative higher CR only with 3,5-Dicaffeoylquinic acid(12.5%). A colloidal gold conjugated anti-luteoloside monoclonal antibody was also prepared and used in an immunochromatographic assay for luteoloside. The limit of detection for the CGICA was found to be around 200~400 ng/m L. The CRs with luteolin-7-O-glucuronide, baicalin and scutellarin were 8 000%, 1 000% and 666%, respectively.The developed ic ELISA and CGIC test strips were used to determinate the content of CGA and luteoloside in FLJ samples and the results were agreed well with those detected by HPLC method. The CGIC test strips were also used for quality control of Chinese patent medicine.Conclusion: The developed ic ELISA was suitable for the detection of CGA and luteoloside with the characterized of simple, rapid, reliable, low costs, and high throughout. Moreover, the usefulness of the CGIC dipsticks as a quality control method was confirmed to be an easy and timely manner with the advantages includeing a short assay time(10 min) and no dependence on any instrumental systems. |
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