Purification and characterization of monoclonal IgG antibodies recognizing Ebola virus glycoprotein

PURPOSE: The goal of this study was to purify and characterize Ebola virus glycoprotein (GP)-specific IgG antibodies from hybridoma clones. MATERIALS AND METHODS: For hybridoma production, mice were injected by intramuscular-electroporation with GP DNA vaccines, and boosted with GP vaccines. The spleen cells were used for producing GP-specific hybridoma. Enzyme-linked immunosorbent assay, Western blot assay, flow cytometry, and virus-neutralizing assay were used to test the ability of monoclonal IgG antibodies to recognize GP and neutralize Ebola virus. RESULTS: Twelve hybridomas, the cell supernatants of which displayed GP-binding activity by enzyme-linked immunosorbent assay and the presence of both IgG heavy and light chains by Western blot assay, were chosen as a possible IgG producer. Among these, five clones (C36-1, D11-3, D12-1, D34-2, and E140-2) were identified to secrete monoclonal IgG antibodies. When the monoclonal IgG antibodies from the 5 clones were tested for their antigen specificity, they recognized GP in an antigen-specific and IgG dose-dependent manner. They remained reactive to GP at the lowest tested concentrations (1.953–7.8 ng/mL). In particular, IgG antibodies from clones D11-3, D12-1, and E140-2 recognized the native forms of GP expressed on the cell surface. These antibodies were identified as IgG1, IgG2a, or IgG2b kappa types and appeared to recognize the native forms of GP, but not the denatured forms of GP, as determined by Western blot assay. Despite their GP-binding activity, none of the IgG antibodies neutralized Ebola virus infection in vitro, suggesting that these antibodies are unable to neutralize Ebola virus infection. CONCLUSION: This study shows that the purified IgG antibodies from 5 clones (C36-1, D11-3, D12-1, D34-2, and E140-2) possess GP-binding activity but not Ebola virus-neutralizing activity.

Preparation and identification of monoclonal antibody against BNTN3 A3 / 军事医学

Objective To prepare and identify monoclonal antibodies against BTN 3A3.Methods BALB/c mice were immunized with the BTN3A3 recombinant protein BTN3A3-mFc containing the mouse IgG2a Fc tag as an antigen, and hybridoma cells were obtained by cell fusion .BTN3A3 recombinant protein BTN3A3-hFc containing a human IgG1 Fc tag as a detection target was used to screen hybridoma cell lines secreting monoclonal antibodies against BTN 3A3.The above monoclonal antibodies were identified with Western blot and indirect immunofluorescence .Results Seven hybridoma cell lines that could stably secret BTN 3A3 monoclonal antibodies were obtained . ELISA, Western blot and indirect immunofluorescence showed that the above antibodies specifically recognized BTN 3A3.Conclusion BTN3A3 monoclonal antibodies that can be applied to ELISA , Western blot or immunofluorescence have been obtained , which can contribute to the functional study of BTN3A3.

The preparation of the mouse monoclonal antibodies specific for the DNAH2 protein / 天津医药

Objective To prepare specific mouse monoclonal antibodies against Homo sapiens dynein,axonemal, heavy chain 2 (DNAH2). Methods Firstly, recombinant plasmid encoding His tagged immunogen, targeting N-terminal sequence of DNAH2 protein (1-300 aa), in E. coli was constructed. IPTG was used to induce the expression of His-immunogen, which was then purified and immunized in BALB/c mice. Hybridoma cells were obtained through the fusion between myeloma cells and splenocytes isolated from BALB/c mice. Finally, ELISA and Western blot assays were performed to screen the positive hybridoma. Results IPTG was used efficiently to induce the expression of DNAH2 immunogen in E. coli. DNAH2 protein bands were detected in screened positive hybridoma. Conclusion Mouse monoclonal anti-DNAH2 antibody is prepared successfully.

New Monoclonal Antibodies to Human IgA: Obtaining and Study of Biological Properties.

Aim: Preparation of monoclonal antibodies to human IgA, investigation of their properties and selection of the most appropriate McAbs for highly sensitive and specific immunoassay tests. Methodology: Balb/c mice were used for monoclonal antibodies (McAbs) production. Animals were immunized subcutaneously with purified preparation of human IgA. Immunization duration - 7 days, B cells source - regional lymph nodes. Hybridization of immunocompetent cells and myeloma cells was performed with polyethylene glycol as a fusogen. Screening and subsequent hybridoma clones selection was performed by ELISA-test using human IgA and IgA Fc-fragments, IgG and human IgM. To determine the isotype of McAbs, titer, affinity constants, and to identify its comparative epitopic specificity appropriate modifications of ELISA-test were used. Results: In our experiments new hybrid clones selection scheme to define the most appropriate McAbs for highly sensitive and specific immunoassay tests was developed. It was established that the most prospective were hybridoma clones producing antibodies against Fc-region of IgA molecule. It was proposed to have a comprehensive description of antibodies, which included the establishment of its isotype, titer, and affinity constants. In view of the further use of obtained McAbs for development of highly informative immunoassay methods, only high titer and affinity antibodies were selected. Since IgM-antibodies are characterized by low specificity and affinity, McAbs of such isotype have not been chosen for further study. Focusing on more efficient McAbs purification on protein A based sorbents, antibodies with IgG2a, IgG2b, and IgG3 isotypes were selected. It was established that there is a correlation between the antibody titer in the culture fluid and its affinity constant: antibodies with titer more than 1:500 characterized by an affinity constant not less than 109 М-1. Conclusion: A set of 14 new monoclonal antibodies to human IgA has been obtained. Following biological properties of obtained McAbs has been studied: Activity in the indirect ELISA, specificity within IgA molecule (Fab- or Fc- fragment), cross-reactivity with other classes of serum immunoglobulins (human IgG, and IgM), titer in the culture fluid, affinity constant, and relative epitope specificity. Criteria of McAbs selection for their further use in highly sensitive and specific immunoassay methods have been developed. McAbs with following characteristics are the most promising for these purposes: they should be directed to the Fc-fragment of IgA, have a high signal in the indirect ELISA, absence of cross-reactivity with other classes of immunoglobulins, titer in the culture fluid not less then 1:500, and affinity constant not less then 8.0×109 M-1. McAbs of IgM isotype are not suitable for effective biotechnological approaches.

Preparation and Preliminary Application of Monoclonal Antibody Against Carcinoembryonic Antigen Glypican-3 / 天津医药

Objective To prepare monoclonal antibody specifically against carcinoembryonic antigen glypican-3 (GPC3) and its preliminary application. Methods GPC3 was cloned with PCR to pET16b vector and expressed in E. co-liBL21. Spleen cells were obtained from Balb/c mice embedding immunized with purified antigen intraperitoneally, and fused with Sp2/0 cells. Hybridoma cells were screened by indirect ELISA, and identified by Western blot assay using puri-fied protein after the cell fusion. The indirect immunofluorescence method was used to detect the GPC3 expression in HepG2 cell line. Results The prokaryotic expression vector of GPC3 was successfully constructed, and GPC3 was stably expressed in E. coliBL21. A mouse hybridoma cell line secreting monoclonal antibody to GPC3 was obtained. Western blot analysis showed that monoclonal antibody specifically recognized recombinant protein. Monoclonal antibody could be used to detect GPC3 protein expression in HepG2 cell line by indirect immunofluorescence. Conclusion The monoclonal antibody against GPC3 was successfully obtained.

Obtención de anticuerpos monoclonales de ratón contra proteasa de cisteína 5 recombinante de Entamoeba histolytica / Production of monoclonal antibodies against cysteine protease 5 of Entamoeba histolytica

Objetivo. Obtener anticuerpos monoclonales de ratón contra la proteasas de cisteína 5 (EhCP5) de Entamoeba histolytica. Materiales y métodos. Se inmunizaron ratones BALB/c por vía intraperitoneal con adyuvante de Freund completo e incompleto con la proteína recombinante EhCP5 obtenida a partir del cultivo de E.coli DH5α trasfectada con el vector recombinante pJC45 que expresa dicha proteína. Se seleccionó el animal con mejor respuesta de anticuerpos. Al cual se le extrajo su bazo como fuente de linfocitos B, los cuales se fusionaron utilizando PEG con células de mieloma de ratón SP2-0/Ag14. Se procedió a selección de los hibridomas y a la evaluación de los sobrenadantes de las colonias que crecieron a los 7 días mediante ELISA. Los hibridomas con valores más altos de anticuerpos específicos contra la proteína EhCP5r se seleccionaron, y los clones obtenidos por diluciones limitantes fueron expandidos. Resultados. A partir de un clon secretor estable se purifico el anticuerpo monoclonal anti EhCP5r del isotipo IgG1 por cromatografía de afinidad con proteína G. Los clones fueron expandidos in vivo ein vitro. Con el anticuerpo purificado se diseñaron tres sistemas de captura para evaluar la aplicabilidad del anticuerpo monoclonal anti EhCP5r como método inmunodiagnóstico. Conclusiones. Se logro la producción de un anticuerpo monoclonal específico contra EhCP5r que permite diferenciar Entamoeba histolytica de Entamoeba dispar.

Objective. Obtain mouse monoclonal antibodies against cysteine proteases 5 (EhCP5) of Entamoeba histolytica. Materials and methods. BALB/c mice were immunized intraperitoneally with complete and incomplete Freund adjuvant EhCP5 with the recombinant EhCP5 protein obtained from E.coli DH5α culture transfected with the recombinant vector pJC45 that expresses said protein. The animal with the best antibody response was selected. Its spleen was extracted as a source of B-lymphocytes, which were merged using PEG miceSP2-0/Ag14 myeloma cells. The team proceeded to undergo the selection of the hybridomas and the evaluation of the supernatants of the colonies that grew after 7 days by ELISA. The hybridomas with higher values of specific antibodies against the protein EhCP5r were selected, and clones obtained by limiting dilution were expanded Results. With the use of a stable secreting clone the monoclonal antibody anti EhCP5r IgG1 isotype was purified by affinity chromatography with protein G. The clones were expanded in vivo and in vitro. Three capture systems were designed with the purified antibody to assess the applicability of the monoclonal antibody anti EhCP5r as an immunodiagnostic method. Conclusions. The production of a specific monoclonal antibody against EhCP5r was achieved to differentiate Entamoeba histolytica from Entamoeba dispar.

Progress in research on preparation of antibodies against rhesus D antigen / 军事医学科学院院刊

The RhD antigen is expressed only in human red blood cells (RBC).Its immunogenicity and clinical application are only next to ABO blood group system, and is widely used both for blood typing and prevention of hemolytic disease of the newborn. The traditional anti-Rh(D) is derived by fractionation of plasma from individuals who have been sensitized by pregnancy or transfusion, or have been deliberately immunized to produce anti-Rh(D). Because of the limited source of plasma, researchers began the study of monoclonal and recombinant antibody. Monoclonal and recombinant anti-Rh(D) antibodies may provide alternatives to the current plasma derived polyclonal IgG anti-Rh(D), but up to now,none of them have yet proved effective in humans for prevention of RhD immunity and hemolytic disease of the newborn.

Preparation and identification of monoclonal antibodies against human programmed cell death 10(PDCD10) / 北京大学学报(医学版)

Objective: To obtain monoclonal antibodies against programmed cell death 10(PDCD10) for further study of the structure and function of PDCD10 protein.Methods: Balb/c mice were immunized with recombinant PDCD10,hybridoma cell lines secreting monoclonal antibodies against PDCD10 were screened by regular cell fusion and subcloning approach.The specificities of these monoclonal antibodies were determined by ELISA,Western blotting and Immunofluorescecence assay.Results: Three hybridoma cell lines(5G1,4F7 and 3H5) stable in secreting specific monoclonal antibodies were successfully obtained.Subclass of IgG belonged to IgG1(4F7 and 5G1)and IgG2b(3H5),respectively.The ascite titers of these monoclonal antibodies reached 1∶10~7.They could specifically bind to recombinant PDCD10 and endogenous and overexpressed PDCD10 proteins proved by ELISA and Western blotting.They failed to react with E.coli lysates and glutathione S-transferase(GST).In addition,these three monoclonal antibodies could recognize different epitopes of PDCD10 proteins assessed by immune fluorescence competitive binding assay.Both endogenous and overexpressed PDCD10 protein mainly located in the nucleus.Conclusion: Monoclonal antibodies against PDCD10 with high titers and specificity have been successfully prepared,which has laid the foundation for further study of PDCD10 protein.

Cloning of survivin gene and preparation its monoclonal antibodies as well as checking survivin expresson in liver carcinoma cells / 中华检验医学杂志

Objective To clone survivin gene, prepare its monoclonal antibody(McAb) and check its expression in liver carcinoma cells.Methods Survivin gene cDNA was amplified by RT-PCR from human breast carcinoma cell line MCF-7 and constructed into prokaryocytic expression vector.Fusion-protein of human Survivin was expressed and used for immunizing BALB/C mice.The spleen cells from immunized mice were fused with SP2/0 cells and selectively cultured with HAT medium.ELISA and Western blot were used to screen and identify the McAbs.Immunocytochemical staining was applied for survivin expression in liver carcinoma cells.Results The full survivin gene was cloned. The hybridoma cell that secret specific monoclonal antibody against human surviving was identified.The immunoglobulin subclasses of the McAbs were IgG1.Western blot showed that the McAbs against survivin can specifically react with MS-Survivin fusion protein. The positive reaction was found in hepatocarcinoma cell line HepG2 and hepatocarcinoma tissue by immunocytochemical staining.Conclusion The McAbs against the human Survivin were successfully prepared by a MS2-Survivin fusion protein expressed by E.coli. and the McAb had positive reaction with HepG2 and hepatocarcinoma tissue. It may be a useful tool to study the functions of survivin and check clinical cancer samples.

Preparation and identification of monoclonal antibody against angiopoietin-related protein 2 / 第二军医大学学报

Objective:To prepare a monoclonal antibody against human angiopoietin-related protein 2(ARP2).Methods: Human spleen ARP2 gene was obtained by RT-PCR.A pET32a-ARP2 plasmid was constructed and was incorporated into(E.coli.) The products were purified and were used to immunize 6-week-old BALB/c female mice.Hybridoma secreting anti-ARP2 monoclonal antibody was obtained by standard procedure.Mass production was carried out after specificity identification with Western blotting.Results: The fusion protein obtained by pET32a system had a relative molecular weight of about 570 000,which was in accordance with the theoretical value.The purity of the protein was more than 90% after purification.The antibody titer was 110~(4)in the hybridoma culture supernatant and 110~(7)-10~(8) in the ascites.The IgG2a type antibody had a relative molecular weight of about 570 000 by Western blot analysis.Immunohistochemistry method showed that the antibody bond with human ARP2.Conclusion: The prepared anti-human ARP2 monoclonal antibody in this study can be used for identification of ARP2 protein.

Estabishment and characterization of mouse hybridoma cell lines secreting monoclonal antibody against annexin B1 / 第二军医大学学报

Objective:To prepare monoclonal antibody against Annexin B for further studying the physiological function of Annexin B1.Methods: Annexin B1 was expressed and purified in E.coli and its antigenicity was confirmed.The product was used to immunize BALB/c mice by intrasplenic embedding.Spleen cells of BALB/c mice were obtained and fused with myeloma cell line SP2/0 in a HAT medium containing 50% PEG 4000 and were then subjected to incubation in 37℃,5% CO_(2)incubator for several days.ELISA was used to assay the type and titer of the McAbs in the supernatant.Results: A hybridoma cell line 2B10H5,which can steadily secrete specific McAbs,was obtained.Conclusion: The McAbs of Annexin B1 have been successfully prepared,which pave a way for further investigation of Annexin B1.

Toxoplasma gondii: caracterización de un anticuerpo monoclonal anti-P30

Se caracterizó un anticuerpo monoclonal específico a Toxoplasma gondii. El hibridoma produjo inmunoglobulinas IgG. El análisis por Western Blot demostró que el anticuerpo monoclonal fue específico para el antígeno de masa molecular aparente de 30 kd, presente en la superficie del parásito. El anticuerpo monoclonal se purificó a partir de fluido ascítico de ratón y se acopló a Sefarosa 4B. Este inmunoabsorbente fue utilizado con el fin de purificar el antígeno parasitario específico. El anticuerpo monoclonal estudiado puede ser de utilidad para las técnicas que contribuyan con el diagnóstico de la toxoplasmosis.

A specific monoclonal antibody was characterized to Toxoplasma gondii. The hybridoma produced IgG immunoglobulins. The Western Blot analysis showed that the monoclonal antibody was specific for the antigen of an apparent mollecular mass of 30 kd, which was present on the antigen surface. The monoclonal antibody was purified starting from mouse´s ascitic fluid and it was matched with Sepharose 4B. This immunoabsorbent was used to purify the specific parasitary antigen. The monoclonal antibody studied may be useful for those tecniques contributing to the toxoplasmosis diagnosis.

Preparation and Characterization of Hybridoma Strains Secreting Natural IgM Against Keratin / 中华皮肤科杂志

Objective To prepare natural anti-keratin IgM monoclonal antibody. Methods Spleen cells of BALB/c mice raised in specific pathogen free conditions were directly fused with Sp2/0 myeloma cells. The hybridoma supernatants were tested by ELISA using pre-extracted keratin. The natural IgM obtained was further identified by immunochemistry and immunoblot methods. Results The cell fusion rate was about 60% without pre-immunization. About 14% supernatants reacted with the keratin antigen. Three hybridoma strains secreting natural IgM monoclonal antibody against keratin were obtained. The immunochemistry results showed that the natural anti-keratin IgM was able to bind to epidermis, sebaceous gland, hair follicule, and muscle tissues. Conclusion B lymphocytes in normal BALB/c mice spleen can produce natural antibody against kerain.

Preparation and analysis of immunological property of monoclonal antibodies against human apolipoprotein E / 中国病理生理杂志

Apolipoprotein E (ApoE) was purified from human serum and used as the antigen to immunize BALB/C mice. The splenic cells of the mice fused with NS-1 mouse myeloma cells. Three hybridoma cell lines(E3C3, E6C3 and E4C2)secreting anti-human ApoE McAb were established. The immunological property of the McAb was studied. The ascites fluids titer were 8?10~(-5)2?10~(-6). The McAbs did not cross react with the other apolipoprotins. ApoE was purified by immunoaffinity chromatography prepared by using the ApoE McAb. McAbs recognized two distinct respective epitopcs on ApoE. The subclass of immunoglabulin of three McAbs were IgG_1. A double McAb sandwich EL1SA was developed to evaluate ApoE levels in normal and hyperlipidemia human serum.

Monoclonal anti-DNA autoantibodies and their idiotypiccross-reactivity in anti-idiotype serum / 中国病理生理杂志

A series of hybridoma cell lines producing monoclonal antibodies to DNA had been obtained by hybridization of spleen cells from MRL-1Pr/1pr mice, which had developed a severe form of SLE and spontaneously produced high levels of antibodies to DNA without exogenus immunization, with SP_2/0-Ag-14 plasmacytomas. The McAb H241, one of those monoclonal antibodies, was used to immunize NZW rabbit and an anti-H241 idiotypic antibody was harvested from immune serum of the rabbit successfully. The sensitivity of H241 and idiotypic cross-reactivity among H241 and other 24 monoclonal anti-DNA autoantibodies in reaction with H241 and anti-H241 idiotypic sera were investigated by radioimmunoassay (RIA). The results demonstrated that H211 had a high sensitivity, 6ng of unlabled H241 was required to achieve 50% inhibition of binding of labeled H241 to anti-H241 idiotypic serum. The experiments also demonstrated that 15 of 24 other monoclonal anti-DNA antibodies represented idiotypic cross-reactivity with H241 in different degrees. These results suggest that although frequently idiotypes have diversity, some antibodies on the basis with similar structure of the idiotypic determinants were limited in the extent of diversity. It is possible that the lupus mouse embryo genes controlling immunoglobuling synthesis involve correlatative loci for some anti-DNA autoantibodies.

Stability of monoclonal antibody-secreting capacity of human-human hybridomas / 中国免疫学杂志

The stability of monoclonal antibody-secreting capacity of three human-h uman hybridomas was studied. It was noticed that there were some differences in the morphology, the size, the number of chromosomes and the secreting-capacity of human-human hybrid cells during expanding culture after cloning or for a long lime culture in vitro. The results showed that the stability of human monoclonal antibody-secreting was concerned with loss of some chromosomes or overgrowth of non-secreting population. The number of tetraploid cells was gradually increased after reselection using HAT medium to these different hybridomas. Meanwhile, some of cells which lost certain chromsomes died quickly. The capacity of monoclonal antibody secreting was recovered in some cell lines. It is important to clone the hybrid cells in time or reselect by using HAT medium when expanding the cloned cells.

Preparation and identification of the monoclonal antibody against human PSMA7 antigen / 解放军医学杂志

Objective To prepare the PSMA7 hybridoma cell lines using classical hybridoma technique and to perform purification and elementary identification of the monoclonal antibodies(McAbs)against PSMA7 antigen for further study.Methods BALB/c mice were immunized with purified recombinant PSMA7 protein.Once the antibody titer of blood taken from mouse tail reached 1∶6 400,a cell fusion was performed between mouse splenic cells and myeloma cells(Sp2/0),and then the hybridoma cell lines secreting monoclonal antibodies against PSMA7 antigen were screened by indirect enzyme linked immunosorbent assay(ELISA).The immunoglobulin subtypes and titer of the monoclonal antibodies against PSMA7 antigen were identified and measured by Western blot analysis and enzyme linked immunosorbent assay,respectively.Results After cell fusion and subcloning,two hybridoma cell lines that stably secreted monoclonal antibodies against PSMA7 antigen were successfully obtained:B013 and B001,which belong to the subtypes of IgG1.The antibody titers in the hybridoma culture supernatant were 1∶128 and 1∶256,and those in the ascites fluid were 1∶25 600 and 1∶32 000,respectively.Western blot analysis and immunohistochemistry showed that the two antibodies can specifically bind with PSMA7 antigen derived from human eucaryotic cells or tissue.Conclusions Two monoclonal antibodies against PSMA7 antigen with high titers and specificity have been successfully prepared.These antibodies can be used for the identification of PSMA7 protein,and may be a useful tool for studying the biological properties of PSMA7 protein.