To purify Methamidophos (Met) monoclonal antibodies with two methods and compare immune activity of purified antibodies. Method Caprylic acid ammonium sulphate precipition (CAASP) method and Sepharose protein-A (S...To purify Methamidophos (Met) monoclonal antibodies with two methods and compare immune activity of purified antibodies. Method Caprylic acid ammonium sulphate precipition (CAASP) method and Sepharose protein-A (SPA) affinity chromatography method were used to purify Met monoclonal antibodies, UV spectrum scanning was used to determine protein content and recovery of purified antibodies, sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the purity of purified antibodies, and enzyme-linked immunosorbent assay (ELISA) was used to determine immune activity of purified antibodies. Results Antibody protein content and recovery rate with CAASP method were 7.62 mg/mL and 8.05% respectively, antibody protein content and recovery rate with SPA method were 6.45 mg/mL and 5.52% respectively. Purity of antibodies purified by SPA method was higher than that by CAASP method. The half-maximal inhibition concentration (IC50) of antibodies purified by SPA to Met was 181.26 靏/mL, and the linear working range and the limit of quantification (LOD) were 2.43-3896.01 靏/mL and 1.03 靏/mL, respectively. The IC50 of antibodies purified by CAASP to Met was 352.82 靏/mL, and the linear working range and LOD were 10.91-11412.29 靏/mL and 3.42 靏/mL, respectively. Conclusion Antibodies purified by SPA method are better than those by CAASP method, and Met monoclonal antibodies purified by SPA method can be used to prepare gold-labelled testing paper for analyzing Met residue in vegetable and drink water.展开更多
Monoclonal antibody (McAb) 3Hll against gastriccancer was grown in the mouse ascites system. Toacquire a clinical grade product for cancer radioimmuno-imaging was purified by two step highperformance liquid chromatogr...Monoclonal antibody (McAb) 3Hll against gastriccancer was grown in the mouse ascites system. Toacquire a clinical grade product for cancer radioimmuno-imaging was purified by two step highperformance liquid chromatography (HPLC) protocolusing protein A and high-performance hydroxylapatite(HPHT). An analysis of data reported shows the twostep HPLC method to be the best purificationprocedure. This protocol satisfies purity and immunoreactivity requirement, and provides an sample sterility,free-pyrogens, free-mycoplasma and non-specific IgGcontamination. This procedure described was capable ofgenerating large amounts of clinical grade monoclonalantibody.展开更多
A new way for the synthesis of human interferon—α_A monoclonal antibody (IFN-α_A-McAb) bound to silica gel packing material in high-performance affinity chromatography (HPAFC) has been developed. The high coupling ...A new way for the synthesis of human interferon—α_A monoclonal antibody (IFN-α_A-McAb) bound to silica gel packing material in high-performance affinity chromatography (HPAFC) has been developed. The high coupling efficiency and specific activity of IFN—α_A-McAb can be obtained by activated diol-silica gel with activating agent. After purification using this packing material in HPAFC, the specific activity of recombinant human interferon-α_A (rIFN-α_A) rose up to 1.03×10~7IU/mg protein and the purification efficiency is appoximately 100 times.展开更多
Chlamydia trachomatis outer membrane protein 2 （Ctomp2） is a major immunogen in chlamydial infections and a highly genus-conserved structural protein of all Chlamydia species. To purify the protein and to prepare mo...Chlamydia trachomatis outer membrane protein 2 （Ctomp2） is a major immunogen in chlamydial infections and a highly genus-conserved structural protein of all Chlamydia species. To purify the protein and to prepare monoclonal antibodies （mAbs） against it, the recombinant protein was induced by IPTG, which was confirmed by SDS-PAGE and purified by means of a Ni^2＋ -charged resin colunm. The denatured protein was refolded in the GSH-GSSH buffer gradually and identified by Westem blotting. Then the BALB/c mice were immunized with the recombinant protein to prepare the mAb against Ctomp2. The obtained mAbs were characterized. Genital specimens were tested with indirect ELISA mostly made of the mAb and cell culture in 84 patients with genital symptoms. The results showed that high-level expression of the recombinant protein was achieved, which existed as inclusion body and amounted to 38 % of total bacterium protein. A mAb against Ctomp2 was obtained. It belongs to IgG 2b. The titers were as high as 1：40 000. The Western blotting showed that the mAb only reacted with the recombinant protein. It had no crossing reactions against E. coli, N. gonorhoea, M. hominis, U. urealyticum and M. penetrans. It had high specifity. In comparison with gold standard test-ceil culture, the sensitivities, specificities, positive predictive values and negative predictive values of indirect ELISA were 95.24%, 100%, 100% and 98.44%, respectively. The above-mentioned research work contributed not only to the further study of the structure and function of this protein, but also to the establishment of the method for its clinical application, for it had not been reported before.展开更多
基金National Natural Science Foundation of China (39970519) Guangxi Basic Studying Fund (0236053) +1 种基金 Guangdong Province Natural Science Foundation (990683) Core Teacher Project of Ministry of Education and Chinese Postdoctoral Fu
文摘To purify Methamidophos (Met) monoclonal antibodies with two methods and compare immune activity of purified antibodies. Method Caprylic acid ammonium sulphate precipition (CAASP) method and Sepharose protein-A (SPA) affinity chromatography method were used to purify Met monoclonal antibodies, UV spectrum scanning was used to determine protein content and recovery of purified antibodies, sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the purity of purified antibodies, and enzyme-linked immunosorbent assay (ELISA) was used to determine immune activity of purified antibodies. Results Antibody protein content and recovery rate with CAASP method were 7.62 mg/mL and 8.05% respectively, antibody protein content and recovery rate with SPA method were 6.45 mg/mL and 5.52% respectively. Purity of antibodies purified by SPA method was higher than that by CAASP method. The half-maximal inhibition concentration (IC50) of antibodies purified by SPA to Met was 181.26 靏/mL, and the linear working range and the limit of quantification (LOD) were 2.43-3896.01 靏/mL and 1.03 靏/mL, respectively. The IC50 of antibodies purified by CAASP to Met was 352.82 靏/mL, and the linear working range and LOD were 10.91-11412.29 靏/mL and 3.42 靏/mL, respectively. Conclusion Antibodies purified by SPA method are better than those by CAASP method, and Met monoclonal antibodies purified by SPA method can be used to prepare gold-labelled testing paper for analyzing Met residue in vegetable and drink water.
文摘Monoclonal antibody (McAb) 3Hll against gastriccancer was grown in the mouse ascites system. Toacquire a clinical grade product for cancer radioimmuno-imaging was purified by two step highperformance liquid chromatography (HPLC) protocolusing protein A and high-performance hydroxylapatite(HPHT). An analysis of data reported shows the twostep HPLC method to be the best purificationprocedure. This protocol satisfies purity and immunoreactivity requirement, and provides an sample sterility,free-pyrogens, free-mycoplasma and non-specific IgGcontamination. This procedure described was capable ofgenerating large amounts of clinical grade monoclonalantibody.
文摘A new way for the synthesis of human interferon—α_A monoclonal antibody (IFN-α_A-McAb) bound to silica gel packing material in high-performance affinity chromatography (HPAFC) has been developed. The high coupling efficiency and specific activity of IFN—α_A-McAb can be obtained by activated diol-silica gel with activating agent. After purification using this packing material in HPAFC, the specific activity of recombinant human interferon-α_A (rIFN-α_A) rose up to 1.03×10~7IU/mg protein and the purification efficiency is appoximately 100 times.
文摘Chlamydia trachomatis outer membrane protein 2 （Ctomp2） is a major immunogen in chlamydial infections and a highly genus-conserved structural protein of all Chlamydia species. To purify the protein and to prepare monoclonal antibodies （mAbs） against it, the recombinant protein was induced by IPTG, which was confirmed by SDS-PAGE and purified by means of a Ni^2＋ -charged resin colunm. The denatured protein was refolded in the GSH-GSSH buffer gradually and identified by Westem blotting. Then the BALB/c mice were immunized with the recombinant protein to prepare the mAb against Ctomp2. The obtained mAbs were characterized. Genital specimens were tested with indirect ELISA mostly made of the mAb and cell culture in 84 patients with genital symptoms. The results showed that high-level expression of the recombinant protein was achieved, which existed as inclusion body and amounted to 38 % of total bacterium protein. A mAb against Ctomp2 was obtained. It belongs to IgG 2b. The titers were as high as 1：40 000. The Western blotting showed that the mAb only reacted with the recombinant protein. It had no crossing reactions against E. coli, N. gonorhoea, M. hominis, U. urealyticum and M. penetrans. It had high specifity. In comparison with gold standard test-ceil culture, the sensitivities, specificities, positive predictive values and negative predictive values of indirect ELISA were 95.24%, 100%, 100% and 98.44%, respectively. The above-mentioned research work contributed not only to the further study of the structure and function of this protein, but also to the establishment of the method for its clinical application, for it had not been reported before.
