Development And Application Of Rapid Detection Kit For Baicalin

Baicalin (baicalein-7-glucuronide), as one of the most important contents purified from Scutellaria baicalensis Georgi., has been used to control the quality of Radix Scutellariae in the Chinese pharmacopoeia. The efficacy of baicalin has been proved in clinical application; therefore, effective measures for the quantitative analysis of baicalin are necessary.Nowadays, the most important techniques available for the quantitative analysis of baicalin can be classified as high-performance liquid chromatography (HPLC), ultra-fast liquid chromatography-tandem mass spectrometry (UPLC-MS) and HPLC-mass spectrometry (HPLC-MS) et al. Although these methods are sensitive and specific, the limination are also exsited such as requiring expensive and complex equipment, highly skilled personnel, relative large amount of samples, and laborious sample pretreatment, which together make it very difficult to screen large numbers of samples in a timely way.To overcome the problems above, a rapid, specific and sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) for detecting baicalin was established and optimized in this study. In addition, a self-made ELISA kit for the rapid quantitative analysis of baicalin was developed based on the above assay. This ELISA kit, based on monoclonal antibodies (MAb) against baicalin (anti-baicalin MAb), has been proved to be highly sensitive and specific in detecting baicalin.The anti-baicalin MAb collected by ascites was purified by caprylic acid precipitation and Protein G column, and the results detected by SDS-PAGE and Bradford showed a high purify. The purified MAb was then applied to the establishment of icELISA for baicalin. Parameters of the icELISA process were evaluated and the final reaction parameters of this self-made ELISA kit based on the anti-baicalin MAb were given as follows:Coating condition showed a duliution of 1:8000 (antigen to CBS), and an incubation at 37℃ for 10 min followed 12h at 4℃.5% skim milk was prefered to blocking the wells for 2 h. PBS was chosen to be work buffer and the work concentration of the MAb was assessed to be 1:40000 duliuted with PBS.60 μL of standard solution and 40 μL of the MAb were considered to be the optimal ratio to the reaction and the optional time was 1 h. The color reaction could be kept for 20 min. Linear range of the standard curve was from 3.95 to 125 ng/mL, a mean recovery of 104.6% was obtained, with RSD lower than 5.4%, repeatability and precision associated with relative standard deviations (RSD) were less than 13%. This self-made ELISA kit could be remained at 2-8 ℃ for at least six months. This self-made ELISA kit was used for concentration measurements of baicalin in various tissues homogenate, the value of R2 in regression function is all above 0.9 and the rate of recoveries were 68.1% -111.3% in different tissue tests, which indicates that ELISA method described was successful in determining the baicalin in biological samples.An optimal indirect competition ELISA was established based on anti-baicalin MAb and a reliable, effective, and accurate self-made ELIS A kit for the quantitative analysis of baicalin was developed, which makes it possible to screen large numbers of samples including baicalin in a timely way.