Methodology on specific knock out of glycyrrhizic acid by immunoaffinity chromatography column / 中草药

Objective: To prepare the glycyrrhizic acid (GA) immunoaffinity chromatography column, which could specifically knock out the GA. Methods: An immunoaffinity chromatography (IAC) column was developed by covalently coupling the anti-GA-MAb to CNBr-activated Sepharose™ 4B. The concentration of GA was detected by HPLC, and the maximum coupling capacity, stability, precision, and accuracy of the IAC column for GA were studied. Results: The maximum capacity of the IAC column for GA was 1.326 11 mg, the precision (RSD) of GA was 0.65%, and the accuracy (RE) was 0.37%. RSD of GA stayed 1.37% during 32 d, which showed a pretty well stability. Conclusion: GA-IAC column could rapidly, effectively, and stably knock out the GA.

Expression and purification of CDNF and preparation of its polyclonal antibodies / 中国免疫学杂志

Objective: To obtain purified and functional CDNF-his recombinant protein and prepare its polyclonal antibodies.Methods:Preparation of recombinant CDNF-his was carried out in HEK 293 T cells with pVR1012-CDNF-his successfully constructed transfected into them.Then,the recombinant protein was purified by Ni-NTA immunoaffinity chromatography.The purity was analyzed by SDS-PAGE and the protein′s identity was tested by Western blot.MTT was used to verify the biological function of the protein purified.New Zealand white rabbits were immunized with purified CDNF-his protein for preparation of polyclonal antibodies.Results:pVR1012-CDNF-his expressed successfully in HEK 293 T cells.The purity of protein was up to more than 90%after purification.MTT showed that CDNF-his was able to protect PC 12 cells from damage by 6-OHDA.The polyclonal antibody was detected at the end of animal immunizing process.Conclusion: A method to express and purify protein using HEK 293T cell and following Ni-NTA immunoaffinity chromatography has been built.CDNF-his with biological activity is obtained based that.Finally, polyclonal antibodies of CDNF were generated successfully.

Human Infections with Liver and Minute Intestinal Flukes in Guangxi, China: Analysis by DNA Sequencing, Ultrasonography, and Immunoaffinity Chromatography

The prevalence of liver and intestinal fluke infections was determined by surveying inhabitants of Hengxuan, Fusui, and Shanglin villages which were known to be endemic for liver flukes in Guangxi, China in May 2010. A total of 718 people were examined for helminth eggs by the Kato-Katz thick smear technique, ultrasonography, immunoaffinity chromatography, and DNA sequencing. The overall egg positive rate was found to be 59.6% (28.0-70.6%) that included mixed infections with liver and intestinal flukes. Cases showing higher than 20,000 eggs per gram of feces (EPG) were detected between 1.3% and 16.2%. Ultrasonographic findings exhibited overall 28.2% (72 of 255 cases) dilatation rate of the intrahepatic bile duct. Clonorchis sinensis infection was detected serologically in 88.3% (38 of 43 cases) among C. sinensis egg positive subjects by the immunoaffinity chromatography using a specific antigen for C. sinensis. For differential diagnosis of the liver and intestinal flukes, more precise PCR and nucleotide sequencing for copro-DNA were performed for 46 egg positive cases. Mixed infections with C. sinensis and Metagonimus yokogawai were detected in 8 of 46 egg positive cases, whereas 29 specimens were positive for Haplorchis taichui. Ultrasonographic findings and immunoaffinity chromatography results showed usefulness, even in a limited way, in figuring out of the liver fluke endemicity.

Micropreparation of a Native PHGPx Protein From Radish Seedlings by Immunoaffinity Chromatography / 生物化学与生物物理进展

Radish phospholipid hydroperoxide glutathione peroxidase (RsPHGPx) was identified as a mitochondrion-targeting PHGPx in previous work. To determine its cleavage site of the targeting peptide, the immunoaffinity chromatography (IAC) purification approach was carried out to isolate the native RsPHGPx protein.Polyclonal antibodies directed against recombinant RsPHGPx were raised in rabbit. Monospecific anti-RsPHGPx antibodies were isolated by means of affinity chromatography using the recombinant RsPHGPx as affinity ligand, and employed in assembling an IAC column. A single-step, highly specific and easy-to-use protocol was developed for purification of the active RsPHGPx protein through the assembled IAC column. Using this approach, a specific protein of the expected molecular size was obtained from the mitochondrial fraction of radish seedlings. Western blot analysis showed that it could be specifically recognized by anti-RsPHGPx antibodies, and an enzyme activity assay indicated that it exhibited significant PHGPx activity, suggesting that the purified protein should be the desired native RsPHGPx. These results will lead to clarification of the targeting peptide and the active mature protein of RsPHGPx and will be helpful to further probe the intracellular localization mechanism and biological fun ction of this plant PHGPx.

A novel strategy for subtracting high-abundance proteins in human milk by immunoaffinity chromatography / 解放军医学杂志

Objective To establish and evaluate a novel method for subtracting high-abundance proteins in order to enrich low abundance proteins in human milk, with the aim of discovering important bioactive factors by proteomics approach in the future. Methods The whole proteins in human milk were used as immunogens to prepare polyclonal antibody, and the high-abundance proteins were subtracted by immunoaffinity chromatography. The effect was subsequently evaluated by immunoblotting. Results Anti-human milk polyclonal antibody was successfully prepared following routine method. After immunoaffinity chromatography analysis, four kinds of high-abundance proteins in human milk were removed. Conclusions These data indicated that the anti-human milk polyclonal antibodies prepared by whole protein samples were available for the removal of high-abundance proteins and enriching low abundance proteins of the samples. Accordingly, this method could also be used in detecting low abundance proteins in sample such as serum.

Preparation of monoclonal antibody against human lung cancer and purification of its antigen by the immunoaffinity chromatography / 中国免疫学杂志

Objective:To produce the mcAb specifically reacting with lung cancer and to purificate its antigen.Methods:The mice were immunized with A549, the mcAb 2B9 was screened by indirect cell ELISA and immunohistochemistry, and its antigen was purificated by immunoaffinity chromatography.Results:A mcAb was obtained, which could react to lung cancer but very little or not to normal lung tissue and other caner tissues, and the antigen of the mcAb was purificated from the cell lysate.Conclusion:A mcAb which can react to lung cancer have been obtained and its antigen was purificated, they may be useful on clinic for diagnosis and prognosis of lung cancer.

Immunoaffinity Chromatography Method for Purification of Human Protein C / 中国输血杂志

A special monoclonal antibody(McAb)to protein C(PC)was prepared,itrevealed the different conformation in the absence and presence of Ca~(++).This McAbwas coupled to Sepharose-4B and the gel was loaded into the column for thepurification of PC.The immobilized antibody was capable of binding PC fromthe plasma in the absence of Ca~(++).This immunopurification resulted in a 12 000-fold after one step proceduce.The specific activity of the PC product ranged be-tween 180-230 U/mg and behaved as two bands(?-PC and?-PC)on nonreducedSDS-PAGE and five bands(?-S-PC,?-S-PC,?-heavy chain,?-heavy chainand light chain)on reduced SDS-PAGE.

PURIFICATION OF HUMAN SPERMATOZOA ANTIGEN HS 63 AND ITS ANTIFERTILITY EFFECT / 解剖学报

Conservative sperm acrosomal antigens that react with a monoclonal sperm antibody against human spermatozoa HS63 were purified by immunoaffinity chromatography from rabbit testes. The purity and the molecular weight of the purified antigens were determined by SDS acrylamide gel electrophoresis. There is only a band on the acrylamide gel. The molecular weight is more than 60000. It is found that the antigen is a glycoprotein with the carbohydrate moiety of 17%. Following successive immunization antisera of high titers were raised and shown to react specifically with antigens on sperm acrosome, but not with any somatic cells as judged. By using mouse in vitro fertilization experiments and sperm penetration assay with zona-free hamster ova, the isoimmune sera from rabbit exhibited high degrees of fertilization inhibition as compared to the control sera. The results suggest that sperm-specific antigen reactive to HS63 may be a good candidates for the development of immunocontraceptive vaccines in humans.