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
From the mouse hybridoma cell line secreting an anti-CD4 monoclonal antibody (McAb), total RNA was prepared. The VH and VL genes were amplified by RT-PCR with family specific primer pairs. The PCR products were cloned...From the mouse hybridoma cell line secreting an anti-CD4 monoclonal antibody (McAb), total RNA was prepared. The VH and VL genes were amplified by RT-PCR with family specific primer pairs. The PCR products were cloned into pGEM-T vectors, then tranfected into JM109. The VH and VL genes were snalyzed by automatic DNA sequencer. According to Kabat classification, the VH and VL genes belong to the mouse ig heavy subgroup Ⅱ(A) and x chain subgroupⅢ, respectively. The VH and VL genes were subcloned into pr1-Expr and Pk Expr respectively, then transfected into XL2-Blue. The VH- Pr1 and VL- pk were trans feeted by electroporation into mouse myeloma cell X63Ag8. 653. The transfectoma cells were selected by G418 screening, and then supernatant of cultured transfectoma were analyzed by ELISA and immunofluorescence techniques.We have acquired transfectoma cells secreting anti-CD4 chimeric antibodies.These chimeric antibodies are able to kill tumor cells specifically in vitro.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
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
Summary: The effects of chimeric anti CD4 human/murine chimeric antibody and murine anti CD4 monoclonal antibody (McAb) on the proliferation induced by anti CD3 McAb, phytohemagglutinin (PHA), IL 2, and allogeneic...Summary: The effects of chimeric anti CD4 human/murine chimeric antibody and murine anti CD4 monoclonal antibody (McAb) on the proliferation induced by anti CD3 McAb, phytohemagglutinin (PHA), IL 2, and allogeneic cells were studied. The results showed that chimeric anti CD4 antibody and murine anti CD4 McAb could inhibit the proliferation induced by the above inducers and the inhibitory effects were related to the dosage of the antibodies.展开更多
IM To clone mouse antihuman gastric cancer mAb(3H11) variable genes and to construct 3H11 humanmouse chimeric antibody.METHODS The entire VH and VL genes of antigastric cancer mAb 3H11 were cloned by RTPCR method ...IM To clone mouse antihuman gastric cancer mAb(3H11) variable genes and to construct 3H11 humanmouse chimeric antibody.METHODS The entire VH and VL genes of antigastric cancer mAb 3H11 were cloned by RTPCR method from 3H11 hybridoma cells, using 5′ primers for leader sequences. The 3H11 VL gene was then inserted into humanmouse chimeric light chain expression vector and transfected into murine Sp2/0 myeloma cells.RESULTS DNA sequence analysis indicated that the cloned genes included the whole leader sequences and the mature Ig variable region encoding sequences. After gene transfection, transient expression of chimeric light chain protein was detected.CONCLUSION DNA sequences and transient expression indicated that the cloned gene was functional. This work laid basis for constructing 3H11 humanmouse chimeric antibody in the future..展开更多
Hepatitis due to hepatitis B virus(HBV)reactivation can be serious and potentially fatal,but is preventable.HBV reactivation is most commonly reported in patients receiving chemotherapy,especially rituximab-containing...Hepatitis due to hepatitis B virus(HBV)reactivation can be serious and potentially fatal,but is preventable.HBV reactivation is most commonly reported in patients receiving chemotherapy,especially rituximab-containing therapy for hematological malignancies and those receiving stem cell transplantation.Patients with inactive and even resolved HBV infection still have persistence of HBV genomes in the liver.The expression of these silent genomes is controlled by the immune system.Suppression or ablation of immune cells,most importantly B cells,may lead to reactivation of seemingly resolved HBV infection.Thus,all patients with hematological malignancies receiving anticancer therapy should be screened for active or resolved HBV infection by blood tests for hepatitis B surface antigen(HBsAg)and antibody to hepatitis B core antigen.Patients found to be positive for HBsAg should be given prophylactic antiviral therapy.For patients with resolved HBV infection,there are two approaches.The first is pre-emptive therapy guided by serial HBV DNA monitoring,and treatment with antiviral therapy as soon as HBV DNA becomes detectable.The second approach is prophy-lactic antiviral therapy,particularly for patients receiving high-risk therapy,especially anti-CD20 monoclonal antibody or hematopoietic stem cell transplantation.Entecavir and tenofovir are the preferred antiviral choices.Many new effective therapies for hematological malignancies have been introduced in the past decade,for example,chimeric antigen receptor(CAR)-T cell therapy,novel monoclonal antibodies,bispecific antibody drug conjugates,and small molecule inhibitors,which may be associated with HBV reactivation.Although there is limited evidence to guide the optimal preventive measures,we recommend antivi-ral prophylaxis in HBsAg-positive patients receiving novel treatments,including Bruton’s tyrosine kinase inhibitors,B-cell lymphoma 2 inhibitors,and CAR-T cell therapy.Further studies are needed to determine the risk of HBV reactivation with these agents and the best prophylactic strategy.展开更多
目的分析5种识别α-突触核蛋白(α-synuclein,α-syn)N端结构域的单克隆抗体1C16、2B8、2P21、3O18和1J6的特异性,为帕金森病(Parkinson s disease,PD)的早期诊断和免疫治疗提供抗体支持。方法体外蛋白重组技术获得人源α-syn蛋白(human...目的分析5种识别α-突触核蛋白(α-synuclein,α-syn)N端结构域的单克隆抗体1C16、2B8、2P21、3O18和1J6的特异性,为帕金森病(Parkinson s disease,PD)的早期诊断和免疫治疗提供抗体支持。方法体外蛋白重组技术获得人源α-syn蛋白(human-α-syn,h-α-syn)、鼠源α-syn蛋白(mouse-α-syn,m-α-syn)、β-syn蛋白、N端人源α-syn蛋白(α-syn/N)和去N端人源α-syn蛋白(α-syn/ΔN)。斑点印迹法鉴定5种单克隆抗体的特异性识别结构域,使用蛋白质印迹技术检测其对变性后的纯蛋白和鼠脑组织的识别情况。结果抗体1C16可以识别非变性的h-α-syn纯蛋白和小鼠脑组织中的变性的h-α-syn蛋白,不识别m-α-syn和β-syn。抗体1J6仅识别非变性的h-α-syn及α-syn/N纯蛋白,不识别变性后的全长α-syn纯蛋白和小鼠脑组织中的变性h-α-syn蛋白。结论筛选出的2种单克隆抗体具有特异性,可为本实验下一步生物标志物的酶联免疫吸附法测定检测和针对α-syn的免疫治疗提供基础。展开更多
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From the mouse hybridoma cell line secreting an anti-CD4 monoclonal antibody (McAb), total RNA was prepared. The VH and VL genes were amplified by RT-PCR with family specific primer pairs. The PCR products were cloned into pGEM-T vectors, then tranfected into JM109. The VH and VL genes were snalyzed by automatic DNA sequencer. According to Kabat classification, the VH and VL genes belong to the mouse ig heavy subgroup Ⅱ(A) and x chain subgroupⅢ, respectively. The VH and VL genes were subcloned into pr1-Expr and Pk Expr respectively, then transfected into XL2-Blue. The VH- Pr1 and VL- pk were trans feeted by electroporation into mouse myeloma cell X63Ag8. 653. The transfectoma cells were selected by G418 screening, and then supernatant of cultured transfectoma were analyzed by ELISA and immunofluorescence techniques.We have acquired transfectoma cells secreting anti-CD4 chimeric antibodies.These chimeric antibodies are able to kill tumor cells specifically in vitro.
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.
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Summary: The effects of chimeric anti CD4 human/murine chimeric antibody and murine anti CD4 monoclonal antibody (McAb) on the proliferation induced by anti CD3 McAb, phytohemagglutinin (PHA), IL 2, and allogeneic cells were studied. The results showed that chimeric anti CD4 antibody and murine anti CD4 McAb could inhibit the proliferation induced by the above inducers and the inhibitory effects were related to the dosage of the antibodies.
文摘IM To clone mouse antihuman gastric cancer mAb(3H11) variable genes and to construct 3H11 humanmouse chimeric antibody.METHODS The entire VH and VL genes of antigastric cancer mAb 3H11 were cloned by RTPCR method from 3H11 hybridoma cells, using 5′ primers for leader sequences. The 3H11 VL gene was then inserted into humanmouse chimeric light chain expression vector and transfected into murine Sp2/0 myeloma cells.RESULTS DNA sequence analysis indicated that the cloned genes included the whole leader sequences and the mature Ig variable region encoding sequences. After gene transfection, transient expression of chimeric light chain protein was detected.CONCLUSION DNA sequences and transient expression indicated that the cloned gene was functional. This work laid basis for constructing 3H11 humanmouse chimeric antibody in the future..
文摘Hepatitis due to hepatitis B virus(HBV)reactivation can be serious and potentially fatal,but is preventable.HBV reactivation is most commonly reported in patients receiving chemotherapy,especially rituximab-containing therapy for hematological malignancies and those receiving stem cell transplantation.Patients with inactive and even resolved HBV infection still have persistence of HBV genomes in the liver.The expression of these silent genomes is controlled by the immune system.Suppression or ablation of immune cells,most importantly B cells,may lead to reactivation of seemingly resolved HBV infection.Thus,all patients with hematological malignancies receiving anticancer therapy should be screened for active or resolved HBV infection by blood tests for hepatitis B surface antigen(HBsAg)and antibody to hepatitis B core antigen.Patients found to be positive for HBsAg should be given prophylactic antiviral therapy.For patients with resolved HBV infection,there are two approaches.The first is pre-emptive therapy guided by serial HBV DNA monitoring,and treatment with antiviral therapy as soon as HBV DNA becomes detectable.The second approach is prophy-lactic antiviral therapy,particularly for patients receiving high-risk therapy,especially anti-CD20 monoclonal antibody or hematopoietic stem cell transplantation.Entecavir and tenofovir are the preferred antiviral choices.Many new effective therapies for hematological malignancies have been introduced in the past decade,for example,chimeric antigen receptor(CAR)-T cell therapy,novel monoclonal antibodies,bispecific antibody drug conjugates,and small molecule inhibitors,which may be associated with HBV reactivation.Although there is limited evidence to guide the optimal preventive measures,we recommend antivi-ral prophylaxis in HBsAg-positive patients receiving novel treatments,including Bruton’s tyrosine kinase inhibitors,B-cell lymphoma 2 inhibitors,and CAR-T cell therapy.Further studies are needed to determine the risk of HBV reactivation with these agents and the best prophylactic strategy.
文摘目的分析5种识别α-突触核蛋白(α-synuclein,α-syn)N端结构域的单克隆抗体1C16、2B8、2P21、3O18和1J6的特异性,为帕金森病(Parkinson s disease,PD)的早期诊断和免疫治疗提供抗体支持。方法体外蛋白重组技术获得人源α-syn蛋白(human-α-syn,h-α-syn)、鼠源α-syn蛋白(mouse-α-syn,m-α-syn)、β-syn蛋白、N端人源α-syn蛋白(α-syn/N)和去N端人源α-syn蛋白(α-syn/ΔN)。斑点印迹法鉴定5种单克隆抗体的特异性识别结构域,使用蛋白质印迹技术检测其对变性后的纯蛋白和鼠脑组织的识别情况。结果抗体1C16可以识别非变性的h-α-syn纯蛋白和小鼠脑组织中的变性的h-α-syn蛋白,不识别m-α-syn和β-syn。抗体1J6仅识别非变性的h-α-syn及α-syn/N纯蛋白,不识别变性后的全长α-syn纯蛋白和小鼠脑组织中的变性h-α-syn蛋白。结论筛选出的2种单克隆抗体具有特异性,可为本实验下一步生物标志物的酶联免疫吸附法测定检测和针对α-syn的免疫治疗提供基础。