To produce the monoclonal antibodies (mAbs) against hygromycin B phosphotransferase (HPT) and to develop immunoassay based on mAbs for biosafety assessment of HPT in genetically modified rice (GM rice). Methods ...To produce the monoclonal antibodies (mAbs) against hygromycin B phosphotransferase (HPT) and to develop immunoassay based on mAbs for biosafety assessment of HPT in genetically modified rice (GM rice). Methods BALB/c mice were immunized with purified recombinant 6His. HPT protein, and the conventional hybridoma technology was used to generate the monoclonal hybridoma cells. ELISA and Western blot were used to analyze the specificity of mAbs recognizing HPT and the cross reaction with other proteins. A double-Ab sandwich ELISA method was established to detect HPT expression level in the sck gene-modified rice plants. Results Four hybridomas, named F1,D4-2, D4-4, and D4-5, producing the mAbs against HPT were successfully obtained with the titer of ascetic mAbs ranging from 10×10^-4 to10×10^-5. Identification of subclass showed that all the produced mAbs belonged to IgG1. Western blot showed specific binding reaction between the mAbs to the HPT proteins expressed in the GM rice. A double sandwich ELISA coated with anti-HPT polyclonal antibody was established with mAbs as sandwich antibody, which showed a sensitivity of 30ng/mL and did not crossreact with other proteins. The expression level of HPT in the leaves of sck-transformed lines was detected (80-150ng/mL). But HPT protein in the grain and seed of GM rice could not be detected using this ELISA assay, Conclusion Anti-HPT mAbs prepared herein have a high specificity and can be used for rapid assay of HPT antigen. The expression level of HPT in the GM rice grain and seed is lower than our ELISA detection limit.展开更多
A new way for the synthesis of human interferon—α;monoclonal antibody (IFN-α;-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;monoclonal antibody (IFN-α;-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;-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;(rIFN-α;) rose up to 1.03×10;IU/mg protein and the purification efficiency is appoximately 100 times.展开更多
Immunoconjugates are antibody-drug hybrid molecules which combine the exquisite selectivity or monoclonal antibodies with the potent toxicity of anticancer agents. A monoclonal antibody SZ39 against human brain glioma...Immunoconjugates are antibody-drug hybrid molecules which combine the exquisite selectivity or monoclonal antibodies with the potent toxicity of anticancer agents. A monoclonal antibody SZ39 against human brain gliomas was used as a drug carrier. Adriamycin (ADR) was bound covalently to SZ39 to form a SZ39-ADR conjugate. The cytotoxic activity of the SZ39-ADR conjugate was tested in vitro and demonstrated potent and specific killing of cells derived from a human malignant glioma. 50% inhibitory concentration (IC50) for SZ39-ADR to 'target' cells was 8.14×10-9 M. An index of specificity between 'target' and 'non-target' cells was calculated to be 88-fold. These data suggest that the SZ39-ADR may use as a potent and cell type-specific agent and is a likely candidate for the targeting chemotherapy of malignant gliotnas.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
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 mon...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 Ni2+ -charged resin column. The denatured protein was refolded in the GSH-GSSH buffer gradually and identified by Western 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-cell 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
HMBOX1 was a novel transcription factor possibly involving in function of pancreas and cytotoxicity of NK cells. For function determination, recombinant human HMBOX1 protein was obtained and purified, and the monoclon...HMBOX1 was a novel transcription factor possibly involving in function of pancreas and cytotoxicity of NK cells. For function determination, recombinant human HMBOX1 protein was obtained and purified, and the monoclonal antibodies against HMBOX1 were prepared. The full-length cDNA fragment encoding HMBOX1 was amplified from NK-92 cells and inserted into prokaryotic expression vector pET22b. The pET22b-HMBOX1-6his vector was then transformed into E. coli Rosetta (DE3) and induced by 1 mM IPTG for 4 h at 37℃. The fusion HMBOX1 protein was mainly expressed in inclusion bodies, which was purified and refolded using Ni^2+-affinity chromatography. With the purified fusion HMBOX1 protein as antigen, monoclonal antibodies against HMBOX1 were generated, providing a potentially useful tool for further study in HMBOX1 functions. Using these anti-HMBOX1 mAbs, we identified that HMBOX1 is located in both cytoplasm and nucleus and could be detected in 10 human normal tissues, including cerebrum, pancreas, kidney and liver tissues. Moreover, the expression in hepatic carcinoma was significantly lower than that in adjacent tissues.展开更多
Objective To prepare and identify monoclonal antibody (McAb) against recombinant human tissue factor pathway inhibitor (rhTFPI) and to use it for measurement of TFPI by ELISA, and to evaluate the effects of the McAb...Objective To prepare and identify monoclonal antibody (McAb) against recombinant human tissue factor pathway inhibitor (rhTFPI) and to use it for measurement of TFPI by ELISA, and to evaluate the effects of the McAb on dilute prothrombin time (PT) and activated partial thromboplastin time (APTT).Methods After intrasplenic immunization of Balb/c mouse with TFPI, hybridoma technique was used to raise monoclonal antibody against rhTFPI. The McAb was wellcharacterized and labelled with horseradish peroxidase (HRP) by using assay of TFPI in ELISA. Furthermore, the McAb was added to normal and factor Ⅸ deficient plasma for observation of dilute PT and APTT.Results Two hybridomas (4F4, 4F8) secreting McAb against TFPI were established. The Ig class and subclass of the McAb purified from 4F8 was IgG1. Immunoblotting results indicated that the McAb4F8 only recognized a single band of TFPI with molecular weight of 34.8 KD. The results of Sandwich enzymelinked immunosorbent assay (ELISA) by using the HRP labelled McAb4F8 showed that the mean of TFPI in normal human plasma is 103.2±11.5 μg/L. The McAb 4F8 was also proved to shorten markedly dilute prothrombin time of factor Ⅸ deficient plasma and normal plasma.Conclusions We established two hybridomas cell lines (4F4, 4F8) and obtained the McAb4F8 against TFPI and reported the levels of TFPI in healthy adult human plasma by Sandwich ELISA with HRP labelled McAb4F8 in Chinese.展开更多
AIM To explore relationships between human carcinomas and mycoplasma infection.METHODS Monoclonal antibody PD4, which specifically recognizes a distinct protein from mycoplasma hyorhinis, was used to detect mycoplasma...AIM To explore relationships between human carcinomas and mycoplasma infection.METHODS Monoclonal antibody PD4, which specifically recognizes a distinct protein from mycoplasma hyorhinis, was used to detect mycoplasma infection in different paraffinembedded carcinoma tissues with immunohistochemistry. PCR was applied to amplify the mycoplasma DNA from the positive samples for confirming immunohistochemistry.RESULTS Fifty of 90 cases (56%) of gastric carcinoma were positive for mycoplasma hyorhinis. In other gastric diseases, the mycoplasma infection ratio was 28% (18/49) in chronic superficial gastritis, 30% (14/ 46) in gastric ulcer and 37% (18/ 49) in intestinal metaplasia. The difference is significant with gastric cancer (X2=12.06, P<0.05). In colon carcinoma, the mycoplasma infection ratio was 55.1% (32/58), but it was 20.9% (10/49) in adenomarous polyp (X2=13.46, P<0.005).Gastric and colon cancers with high differentiation had a higher mycoplasma infection ratio than those with low differentiation (P< 0.05). Mycoplasma infection in esophageal cancer, lung cancer, breast cancer and glioma was 50.9% (27/53), 52.6% (31/ 59), 39.7%(25/63) and 41% (38/91), respectively. The mycoplasma DNA was successfully amplified with the DNA extracted from the cancer tissues that were positive for mycoplasma infection (detected with antibody PD4).CONCLUSION There was high correlation between mycoplasma infection and different cancers, which suggests the possibility of an association between the two. The mechanism involved in oncogenesis by mycoplasma remains unknown.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
基金This work was supported by 973 National Fundamental Science Program Funds (2001CB109007 and 2001CB10901)and 863 National High-Tech Program Funds (2004AA212221 and 2002AA212041).
文摘To produce the monoclonal antibodies (mAbs) against hygromycin B phosphotransferase (HPT) and to develop immunoassay based on mAbs for biosafety assessment of HPT in genetically modified rice (GM rice). Methods BALB/c mice were immunized with purified recombinant 6His. HPT protein, and the conventional hybridoma technology was used to generate the monoclonal hybridoma cells. ELISA and Western blot were used to analyze the specificity of mAbs recognizing HPT and the cross reaction with other proteins. A double-Ab sandwich ELISA method was established to detect HPT expression level in the sck gene-modified rice plants. Results Four hybridomas, named F1,D4-2, D4-4, and D4-5, producing the mAbs against HPT were successfully obtained with the titer of ascetic mAbs ranging from 10×10^-4 to10×10^-5. Identification of subclass showed that all the produced mAbs belonged to IgG1. Western blot showed specific binding reaction between the mAbs to the HPT proteins expressed in the GM rice. A double sandwich ELISA coated with anti-HPT polyclonal antibody was established with mAbs as sandwich antibody, which showed a sensitivity of 30ng/mL and did not crossreact with other proteins. The expression level of HPT in the leaves of sck-transformed lines was detected (80-150ng/mL). But HPT protein in the grain and seed of GM rice could not be detected using this ELISA assay, Conclusion Anti-HPT mAbs prepared herein have a high specificity and can be used for rapid assay of HPT antigen. The expression level of HPT in the GM rice grain and seed is lower than our ELISA detection limit.
文摘A new way for the synthesis of human interferon—α;monoclonal antibody (IFN-α;-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;-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;(rIFN-α;) rose up to 1.03×10;IU/mg protein and the purification efficiency is appoximately 100 times.
文摘Immunoconjugates are antibody-drug hybrid molecules which combine the exquisite selectivity or monoclonal antibodies with the potent toxicity of anticancer agents. A monoclonal antibody SZ39 against human brain gliomas was used as a drug carrier. Adriamycin (ADR) was bound covalently to SZ39 to form a SZ39-ADR conjugate. The cytotoxic activity of the SZ39-ADR conjugate was tested in vitro and demonstrated potent and specific killing of cells derived from a human malignant glioma. 50% inhibitory concentration (IC50) for SZ39-ADR to 'target' cells was 8.14×10-9 M. An index of specificity between 'target' and 'non-target' cells was calculated to be 88-fold. These data suggest that the SZ39-ADR may use as a potent and cell type-specific agent and is a likely candidate for the targeting chemotherapy of malignant gliotnas.
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 Ni2+ -charged resin column. The denatured protein was refolded in the GSH-GSSH buffer gradually and identified by Western 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-cell 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Acknowledgements This work was supported by grants from National Natural Science Foundation of China (#30671901 #30628014), National 863 Science Program by Ministry of Science and Technology of China (#2007AA021010, 2007AA021109), National 973 Basic Science Project by Ministry of Science & Technology of China (#2010CB911901).
文摘HMBOX1 was a novel transcription factor possibly involving in function of pancreas and cytotoxicity of NK cells. For function determination, recombinant human HMBOX1 protein was obtained and purified, and the monoclonal antibodies against HMBOX1 were prepared. The full-length cDNA fragment encoding HMBOX1 was amplified from NK-92 cells and inserted into prokaryotic expression vector pET22b. The pET22b-HMBOX1-6his vector was then transformed into E. coli Rosetta (DE3) and induced by 1 mM IPTG for 4 h at 37℃. The fusion HMBOX1 protein was mainly expressed in inclusion bodies, which was purified and refolded using Ni^2+-affinity chromatography. With the purified fusion HMBOX1 protein as antigen, monoclonal antibodies against HMBOX1 were generated, providing a potentially useful tool for further study in HMBOX1 functions. Using these anti-HMBOX1 mAbs, we identified that HMBOX1 is located in both cytoplasm and nucleus and could be detected in 10 human normal tissues, including cerebrum, pancreas, kidney and liver tissues. Moreover, the expression in hepatic carcinoma was significantly lower than that in adjacent tissues.
文摘Objective To prepare and identify monoclonal antibody (McAb) against recombinant human tissue factor pathway inhibitor (rhTFPI) and to use it for measurement of TFPI by ELISA, and to evaluate the effects of the McAb on dilute prothrombin time (PT) and activated partial thromboplastin time (APTT).Methods After intrasplenic immunization of Balb/c mouse with TFPI, hybridoma technique was used to raise monoclonal antibody against rhTFPI. The McAb was wellcharacterized and labelled with horseradish peroxidase (HRP) by using assay of TFPI in ELISA. Furthermore, the McAb was added to normal and factor Ⅸ deficient plasma for observation of dilute PT and APTT.Results Two hybridomas (4F4, 4F8) secreting McAb against TFPI were established. The Ig class and subclass of the McAb purified from 4F8 was IgG1. Immunoblotting results indicated that the McAb4F8 only recognized a single band of TFPI with molecular weight of 34.8 KD. The results of Sandwich enzymelinked immunosorbent assay (ELISA) by using the HRP labelled McAb4F8 showed that the mean of TFPI in normal human plasma is 103.2±11.5 μg/L. The McAb 4F8 was also proved to shorten markedly dilute prothrombin time of factor Ⅸ deficient plasma and normal plasma.Conclusions We established two hybridomas cell lines (4F4, 4F8) and obtained the McAb4F8 against TFPI and reported the levels of TFPI in healthy adult human plasma by Sandwich ELISA with HRP labelled McAb4F8 in Chinese.
基金Supported by National 863 Project (102-10-01-08)National Natural Science Foundation of China(39570405)+1 种基金Natural Science Foundation of Beijing(7941001)State Key Basic Research Program(G1998051203)
文摘AIM To explore relationships between human carcinomas and mycoplasma infection.METHODS Monoclonal antibody PD4, which specifically recognizes a distinct protein from mycoplasma hyorhinis, was used to detect mycoplasma infection in different paraffinembedded carcinoma tissues with immunohistochemistry. PCR was applied to amplify the mycoplasma DNA from the positive samples for confirming immunohistochemistry.RESULTS Fifty of 90 cases (56%) of gastric carcinoma were positive for mycoplasma hyorhinis. In other gastric diseases, the mycoplasma infection ratio was 28% (18/49) in chronic superficial gastritis, 30% (14/ 46) in gastric ulcer and 37% (18/ 49) in intestinal metaplasia. The difference is significant with gastric cancer (X2=12.06, P<0.05). In colon carcinoma, the mycoplasma infection ratio was 55.1% (32/58), but it was 20.9% (10/49) in adenomarous polyp (X2=13.46, P<0.005).Gastric and colon cancers with high differentiation had a higher mycoplasma infection ratio than those with low differentiation (P< 0.05). Mycoplasma infection in esophageal cancer, lung cancer, breast cancer and glioma was 50.9% (27/53), 52.6% (31/ 59), 39.7%(25/63) and 41% (38/91), respectively. The mycoplasma DNA was successfully amplified with the DNA extracted from the cancer tissues that were positive for mycoplasma infection (detected with antibody PD4).CONCLUSION There was high correlation between mycoplasma infection and different cancers, which suggests the possibility of an association between the two. The mechanism involved in oncogenesis by mycoplasma remains unknown.
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。