| Objective:Preparation of puerarin monoclonal antibodies, and its application study.1Synthesize puerarin-bovine serum albumin (immunogen) and puerarin poly-lysine (including the original) conjugate and monoclonal antibody preparation puerarin.2Use of puerarin monoclonal antibody to puerarin enzyme-linked immunosorbent assay, inspection accuracy, repeatability, reproducibility and recoveries, and the Determination of puerarin in pharmaceutical and biological samples.3Preparation of puerarin immunoaffinity chromatography column, optimizing the knockout, the elution process parameters, examine gel antibody coupling ratio, column capacity, recovery, the knockout samples fingerprinting.4immunoaffinity chromatography column knockout puerarin, puerarin the role of evaluation in the anti-inflammatory effects of Pueraria use of contrast research methods.5puerarin monoclonal antibody fluorescently labeled puerarin on the cell target of preliminary exploration, the use of human umbilical vein endothelial cells.Methods:Preparation of puerarin monoclonal antibody immunoaffinity chromatography and puerarin knock addition to puerarin efficacy, fluorescence labeling method puerarin on the cell target discovery.1Sodium periodate oxidation synthesis puerarin carrier protein conjugates as a package by the original (puerarin-poly-L-lysine) and immunogen (puerarin-bovine serum albumin), immune serum antibodies, after splenic cell fusion, the positive hybridoma screening, monoclonal after ascites was induced large scale preparation of ascites, bitterness ammonium sulfate precipitation and protein G column purified puerarin monoclonal antibody, and calculate the cross-reactivity to antibodies specific detection.2optimization package is the original concentration, the concentration of an anti-linear detection range of conventional indirect competitive ELISA method puerarin enzyme-linked immunosorbent assay. And examine its accuracy, repeatability, reproducibility and recoveries, pharmaceutical and biological samples Determination of puerarin, compared with conventional HPLC assay results, the mobile phase of40%methanol:60%water, and correlation analysis.3puerarin monoclonal antibody by agarose gel activation, antibody-coupled, closed, wash Miscellaneous balance, column packing and preparation the puerarin immune affinity chromatography. Optimization of the screening sample buffer, knockout buffer and elution buffer and flow rate conditions, by examining the antibody gel coupling ratio, the capacity of the column, the recovery rate evaluation prepared puerarin immunoaffinity chromatography column. Establish the knockout sample fingerprints conditions:Agilent1260the Agilent ZORBAX SBC-18column, the injection volume was10ul flow rate of1mL/min, column temperature25℃, mobile phase was methanol aqueous solution, the gradient of concentration of0-10min. methanol methanol concentration by0-23%,10-30min to maintain23%,30-50min methanol methanol from23-35%,50-60min by35-70%.4puerarin immunoaffinity chromatography knockout kudzu root extract puerarin solution to LPS-induced macrophages to secrete IL-lbeta and NO efficacy by puerarin knockout before and after contrast research, observation of puerarin in Pueraria dialogue interleukin-lbeta and nitric oxide’s role in the inhibitory effect.5fluorescent protein puerarin fluorescently labeled monoclonal antibodies, human umbilical vein endothelial cells blocked for1hour in10%serum, incubated for1hour puerarin1μg/mL fluorescently labeled antibody (1:100dilution) and incubated for half an hour after an inverted fluorescence microscope to observe the fluorescent labeling.Results:After performance testing, puerarin monoclonal antibodies were successfully established by the puerarin enzyme-linked immunosorbent assay and immunoaffinity chromatography and stable performance, high precision, good recovery, repeatability to meet the requirements, preparation, can be usedat the puerarin the pharmacological efficacy studies, fluorescently labeled successful, determine the initial targets of puerarin in human umbilical vein endothelial cells located in the nuclear membrane.1puerarin artificial antigen successfully synthesized by UV detector and calculated the coupling ratio of6:1, were successfully prepared by fusion screening cell lines secrete anti-puerarin monoclonal antibody AA9secreted antibody specificity, In addition to51.8%cross-reactivity with skullcap known, no significant cross-reactivity (cross-reactivity ratios<0.01), with the remaining structural analogues.2puerarin monoclonal antibody successfully established enzyme-linked immunosorbent assay, the original concentration to1microg/mL, the antibody concentration of10ng/mL, linear competitive range of10ng-1.28μg, sensitivity (50%inhibition concentration) of128ng/mL, the linear equation:y=-0.156In (x)+1.2804, R2=0.997. Good accuracy and repeatability, hole difference between the coefficient of variation<3%, the the plate difference between the coefficient of variation<7.5%, the average recovery was101.4%, can be used for sample testing.3Preparation of the puerarin immunoaffinity chromatography, knocking elution conditions were optimized and the sample on the sample and the buffer are knockout preferably distilled water,30%methanol elution buffer preferably, the antibody-coupled was98%, the antibody gel Coupling13.5mg/mL, column capacity13.6mg/mL. And preparation of samples and knock In addition to extract HPLC fingerprint successfully established the puerarin knock division.4knock division evaluation of the role of puerarin in Pueraria anti-inflammatory effect. Will be in the logarithmic growth phase macrophages divided into control group (C), model group (M), administered group:Pueraria lobata solution group (plre), puerarin knockout group (PU-KO-plre) and the the puerarin group of (PU). Cultured macrophages by LPS-induced NO and IL-1beta secretion increased significantly, puerarin significantly inhibited IL-1beta secretion can not be suppressed significantly in the knockout Pueraria on LPS-induced IL-1beta secretion inhibition The puerarin produce.5puerarin after purified by protein G monoclonal antibody to be labeled with a fluorescent protein. Human umbilical vein endothelial cells cultured to logarithmic phase divided into control group (C), the puerarin control group (PU), antibody control group (MAb) labeled antibody puerarin group (P+M), by group do not give a labeled antibody or puerarin fully incubated inverted fluorescence microscope to the control group, puerarin control group labeled antibody group had no characteristic fluorescence puerarin labeled antibody group can be observed to the nuclear membrane There are significant fluorescence.Conclusion:Effect of the sodium periodate synthesis puerarin, puerarin monoclonal antibody with high sensitivity, puerarin can be used for trace analysis more quickly and accurately than conventional HPLC methods, the former approach is more streamlined. Immune affinity chromatography can be used in traditional Chinese medicine extraction or the compound material knockout, with the knock on active substances in herbs or compound addition to the before and after comparison study to prove the true pharmacological effects and the contribution. |
PDF Full Text Request