Preparation of human monoclonal anti-C cell line from peripheral blood B lymphocytes of D--donor / 中国输血杂志

【Objective】 To establish human hybridoma cell lines, secreting monoclonal antibody against antigens of Rh blood system, from a donor with rare D--phenotype. 【Methods】 Peripheral blood B lymphocytes of an O type female donor, lacking C/c/E/e antigens on her erythrocyte, were transformed with Epstein-Barr virus (EBVs). EBVs were harvested from the cultural supernatant of B95-8 cells. The transformed lymphoblastoid cell line (LCL) secreting antibodies to C antigens were picked up and then hybridized with the myeloma SHM-D33 using electric fusion technique. Hybridoma cells were selected by HATD-Ouabain(HOTD)(Hypoxantine, Aminopterin, Thymidine, 2-Deoxycytide, and Ouabain)culture medium, microplate antibody screening and limited dilution subcloning. The monoclonal antibody was assayed by serological test and was confirmed by flow cytometry (FCM). 【Results】 From the cultural supernatant of D--peripheral blood transformed B lymphocytes, 3A6-C6, which agglutinated with R1R1(DCe/DCe)O-type RBCs but not with R2R2(DcE/DcE)O-type RBCs, was screened and preliminarily identified as anti-C. We established a hybridoma cell line secreting anti-C immunoglobulin M from B cells of D--individual successfully after hybridization with SHM-D33 myeloma cells. 【Conclusion】 The study had laid the groundwork for future research and development of human monoclonal antibodies against Rh antigens of RBC in future for diagnosis and screening purpose.

Research progress in the rapid preparation of monoclonal antibodies of mouse hybridoma / 生物工程学报

Mouse hybridoma monoclonal antibody is the most commonly used antibody in immunology because of its stable source, easy preparation in later stage and high yield. The traditional time-consuming and laborious hybridoma preparation technology could not meet the growing market demand. In this paper, we describe the rapid preparation techniques involved in antigen design and screening, B cell enrichment and screening, transgenic myeloma cells, fusion technology improvement, positive hybridoma cell screening and rapid detection of monoclonal antibody performance, to provide a reference for rapid preparation of mouse hybridoma monoclonal antibody.

Preparation of mouse anti-human B7-2 monoclonal antibody and analysis of its role in inducing Fas and FasL on the surface of 8266 cells / 中华微生物学和免疫学杂志

Objective To prepare a mouse anti-human B7-2 monoclonal antibody ( McAb) and to study its effect on the induction of death-related molecules on the surface of tumor cells. -ethods Trans-genic cells, L929-B7-2, were used as the immunogen to immunize BALB/c mice. Through cell fusion, mul-tiple screening by immunofluorescence labeling and continuous subcloning, the hybridoma secreting B7-2 McAb was obtained. Biological characteristics of the McAb were analyzed using Ig subclass identification test strip, antigenic site competition inhibition assay and specific cell membrane molecules binding test. McAb was prepared through inducing ascites in vivo and then purified by protein G affinity chromatography. The purified McAb was co-cultured with 8266 cells, naturally expressing B7-2 molecules, to observe the expres-sion of Fas and FasL on cell surface by flow cytometry ( FCM) . Results The prepared B7-2 McAb labeled as 12G4 was successfully obtained with a titer of 0. 1 μg/5×105 cells. Its heavy and light chains were IgG2b and κ, respectively. The concentration of the purified ascites-derived antibody was 1. 61 mg/ml. FCM re-sults showed that the 12G4 McAb recognized cell membrane molecules well with a positive binding rate of 89. 6％ to 8266 cells. The mean value of the Fas molecule on the cell surface increased after incubating with 20 μg/ml of 12G4 McAb for 12 h and reached the peak of 62575. 8 at 48 h, which was significantly higher than the maximum value of 57135. 4 in the IgG control group (P<0. 05). After culturing the cells with 20μg/ml of 12G4 McAb for 12 h, the expression of FasL on the cell surface also increased and reached the maximum of 7. 98％ at 48 h, which was significantly higher than the 1. 10％ in the IgG control group ( P<0. 05). Conclusions B7-2 McAb was successfully prepared. It could be used to induce the expression of some death-related molecules on the surface of tumor cells.

Preparation of monoclonal antibodies against pneumococcal polysaccharide and hepatitis B virus sur-face protein / 中华微生物学和免疫学杂志

Objective To prepare monoclonal antibodies against pneumonia serotype 33F polysac-charides (Pn33Fps) and hepatitis B virus ( HBV) surface proteins ( HBs) by using the conjugate of Pn33Fps and HBs as antigen. Methods The conjugate of Pn33Fps and HBs was used as antigen to immu-nize mice with different immune doses, different immune procedures and different immune sites. Mouse spleen cells with higher antibody level after immunization were isolated and fused with SP2/0 myeloma cells. The hybridoma cells were screened specifically with Pn33Fps or HBs to prepare corresponding monoclonal antibodies. Results Serum antibodies against Pn33Fps and HBs were induced by immunizing mice with the conjugate. Monoclonal cell lines capable of continuously expressing antibodies against Pn33Fps or HBs were obtained. It has been proved that the recovery rates of samples of Pn33Fps and HBs prepared in three bat-ches tested with ascites monoclonal antibodies prepared by these two monoclonal cell lines were between 95％ and 105％. Conclusions Monoclonal antibodies against Pn33Fps and HBs could be prepared simul-taneously by immunizing mice with the conjugate of Pn33Fps and HBs and used for the quantitative detection of Pn33Fps and HBs.

Preparation of monoclonal antibodies against pneumococcal polysaccharide and hepatitis B virus surface protein / 中华微生物学和免疫学杂志

Objective@#To prepare monoclonal antibodies against pneumonia serotype 33F polysaccharides (Pn33Fps) and hepatitis B virus (HBV) surface proteins (HBs) by using the conjugate of Pn33Fps and HBs as antigen.@*Methods@#The conjugate of Pn33Fps and HBs was used as antigen to immunize mice with different immune doses, different immune procedures and different immune sites. Mouse spleen cells with higher antibody level after immunization were isolated and fused with SP2/0 myeloma cells. The hybridoma cells were screened specifically with Pn33Fps or HBs to prepare corresponding monoclonal antibodies.@*Results@#Serum antibodies against Pn33Fps and HBs were induced by immunizing mice with the conjugate. Monoclonal cell lines capable of continuously expressing antibodies against Pn33Fps or HBs were obtained. It has been proved that the recovery rates of samples of Pn33Fps and HBs prepared in three batches tested with ascites monoclonal antibodies prepared by these two monoclonal cell lines were between 95% and 105%.@*Conclusions@#Monoclonal antibodies against Pn33Fps and HBs could be prepared simultaneously by immunizing mice with the conjugate of Pn33Fps and HBs and used for the quantitative detection of Pn33Fps and HBs.

Production and application of monoclonal antibody against major histocompati-bility complex class Ⅰrelated chain A and B / 中国免疫学杂志

Objective:Generation and identification of hybridoma to produce monoclonal antibody against MICA/B. Methods:SP2/0 cells were fused with murine splenocyte immunized with recombinant protein rMICA?012 to get hybridoma cell lines. The titer of the monoclonal antibody produced by 9B10 cell line was determined by ELISA and its specificity was tested by Western blot,Luminex mutiplex microsphere-based immunoassay and immunofluorescence assay. Results:Six hybridoma cell lines were selected by ELISA screening test. The minimum reaction concentration of mAb 9B10 was 0. 02 ng/μl,and the specificity of mAb 9B10 was determinated by Western blot,Luminex mutiplex microsphere-based immunoassay and immunofluorescence. Conclusion:The monoclonal antibody 9B10 was available to apply for the detection of MICA and MICB expression.

Production and application of monoclonal antibody against major histocompati-bility complex class Ⅰrelated chain A and B / 中国免疫学杂志

Objective:Generation and identification of hybridoma to produce monoclonal antibody against MICA/B. Methods:SP2/0 cells were fused with murine splenocyte immunized with recombinant protein rMICA?012 to get hybridoma cell lines. The titer of the monoclonal antibody produced by 9B10 cell line was determined by ELISA and its specificity was tested by Western blot,Luminex mutiplex microsphere-based immunoassay and immunofluorescence assay. Results:Six hybridoma cell lines were selected by ELISA screening test. The minimum reaction concentration of mAb 9B10 was 0. 02 ng/μl,and the specificity of mAb 9B10 was determinated by Western blot,Luminex mutiplex microsphere-based immunoassay and immunofluorescence. Conclusion:The monoclonal antibody 9B10 was available to apply for the detection of MICA and MICB expression.

Preferential production of IgM-secreting hybridomas by immunization with DNA vaccines coding for Ebola virus glycoprotein: use of protein boosting for IgG-secreting hybridoma production

PURPOSE: The goal of this study was to investigate the utility of DNA vaccines encoding Ebola virus glycoprotein (GP) as a vaccine type for the production of GP-specific hybridomas and antibodies. MATERIALS AND METHODS: DNA vaccines were constructed to express Ebola virus GP. Mice were injected with GP DNA vaccines and their splenocytes were used for hybridoma production. Enzyme-linked immunosorbent assays (ELISAs), limiting dilution subcloning, antibody purification methods, and Western blot assays were used to select GP-specific hybridomas and purify monoclonal antibodies (MAbs) from the hybridoma cells. RESULTS: Twelve hybridomas, the cell supernatants of which displayed GP-binding activity, were selected by ELISA. When purified MAbs from 12 hybridomas were tested for their reactivity to GP, 11 MAbs, except for 1 MAb (from the A6-9 hybridoma) displaying an IgG2a type, were identified as IgM isotypes. Those 11 MAbs failed to recognize GP. However, the MAb from A6-9 recognized the mucin-like region of GP and remained reactive to the antigen at the lowest tested concentration (1.95 ng/mL). This result suggests that IgM-secreting hybridomas are predominantly generated by DNA vaccination. However, boosting with GP resulted in greater production of IgG-secreting hybridomas than GP DNA vaccination alone. CONCLUSION: DNA vaccination may preferentially generate IgM-secreting hybridomas, but boosting with the protein antigen can reverse this propensity. Thus, this protein boosting approach may have implications for the production of IgG-specific hybridomas in the context of the DNA vaccination platform. In addition, the purified monoclonal IgG antibodies may be useful as therapeutic antibodies for controlling Ebola virus infection.

Obtención de sobrenadante de cultivo aglutinador de hematíes portadores del antígeno de grupo sanguíneo B / Obtention of cell culture supernatant for erythrocyte agglutination of blood group B antigen

Introducción: la obtención de anticuerpos monoclonales en líquido ascítico ha ido decayendo paulatinamente por la aparición de alternativas de producción in Vitro, que permiten alcanzar mayores volúmenes y un control más riguroso del proceso productivo, lo que incrementa la reproducibilidad de procesos y la calidad de los productos. Objetivo: evaluar dos métodos de producción de sobrenadante de cultivo rico en una inmunoglobulina de ratón del tipo IgG2b, aglutinadora de hematíes humanos portadores del antígeno del grupo sanguíneo B, según el sistema ABO; la cual es secretada por el hibridoma C6G4. Métodos: se evaluaron dos métodos de producción del anticuerpo con el empleo de un biorreactor CELLine, útil como modelo para la obtención de anticuerpos monoclonales en cultivos de alta densidad celular. Los métodos se diferenciaron esencialmente en la densidad celular de siembra en el biorreactor y en la duración del periodo de fermentación entre la siembra y la cosecha del caldo de cultivo rico en anticuerpos. Para cada método se determinó la concentración específica de anticuerpos y la potencia de aglutinación del sobrenadante, así como la densidad y la viabilidad celular del cultivo alcanzadas en el momento de la cosecha. Resultados: se observó que ambos métodos generaron sobrenadantes de cultivo con una potencia de aglutinación similar, a pesar de que se encontraron diferencias en el resto de las variables medidas. Si bien uno de los métodos produjo una mayor concentración de anticuerpos en el sobrenadante, no se observaron diferencias en la potencia de aglutinación de los sobrenadantes obtenidos por ambas alternativas. Conclusiones: los dos métodos estudiados permitieron obtener volúmenes semejantes de sobrenadante anti-B con diferentes concentraciones de anticuerpos, pero con una potencia de aglutinación similar. La principal diferencia residió en que uno de los métodos permitió obtener el mismo volumen del producto en un tiempo sensiblemente menor...

Introduction : the obtention of monoclonal antibodies in ascite fluid has been declining gradually due to the appearance of alternative in vitro production that achieve higher volumes and a more precise monitoring of the production process, which increases the reproducibility of processes and the quality of products. Objective : to evaluate two methods to make cell culture supernatant rich in murine monoclonal IgG2b type, with agglutinating activity against human red cell of blood group antigen B (ABO system), which is secreted by murine hybridoma C6G4. Methods : two methods were evaluated for antibody production in cell culture supernatant using as model a CELLine bioreactor for the production of monoclonal antibodies in high cell density culture. Both methods essentially differed in the seeding cell density in the bioreactor and the fermentation period between seeding and harvesting of the culture broth rich in antibodies. The specific antibody concentration and potency of agglutination was determined in the obtained supernatant and also the cell density and cell viability of the culture reached at the time of harvest. Results : both methods generated culture supernatants with similar agglutination strength despite differences found in the rest of the variables measured. Even when one of the methods produced a higher antibody concentration in the supernatants, no differences in potency of the supernatants agglutination obtained by both alternatives were observed. Conclusions : both methods generated supernatant anti-B with different concentrations of antibodies but similar potency of agglutination. The main difference was that with one of the methods the same volume of the product was obtained in a considerably minor time...

Specific B-cell Epitope of Per a 1: A Major Allergen of American Cockroach (Periplaneta americana) and Anatomical Localization

PURPOSE: Cockroach (CR) is a common source of indoor allergens, and Per a 1 is a major American CR (Periplaneta americana) allergen; however, several attributes of this protein remain unknown. This study identifies a novel specific B cell epitope and anatomical locations of Per a 1.0105. METHODS: Recombinant Per a 1.0105 (rPer a 1.0105) was used as BALB/c mouse immunogen for the production of monoclonal antibodies (MAb). The MAb specific B cell epitope was identified by determining phage mimotopic peptides and pair-wise alignment of the peptides with the rPer a 1.0105 amino acid sequence. Locations of the Per a 1.0105 in P. americana were investigated by immunohistochemical staining. RESULTS: The rPer a 1.0105 (~13 kDa) had 100%, 98% and > or =90% identity to Per a 1.0105, Per a 1.0101, and Cr-PII, respectively. The B-cell epitope of the Per a 1.0105 specific-MAb was located at residues99 QDLLLQLRDKGV110 contained in all 5 Per a 1.01 isoforms and Per a 1.02. The epitope was analogous to the Bla g 1.02 epitope; however, this B-cell epitope was not an IgE inducer. Per a 1.0105 was found in the midgut and intestinal content of American CR but not in the other organs. The amount of the Per a 1 was ~544 degrees Cg per gram of feces. CONCLUSIONS: The novel Per a 1 B-cell epitope described in this study is a useful target for allergen quantification in samples; however, the specific MAb can be used as an allergen detection reagent. The MAb based-affinity resin can be made for allergen purification, and the so-purified protein can serve as a standard and diagnostic allergen as well as a therapeutic vaccine component. The finding that the Per a 1 is contained in the midgut and feces is useful to increase yield and purity when preparing this allergen.

Specific B-cell Epitope of Per a 1: A Major Allergen of American Cockroach (Periplaneta americana) and Anatomical Localization

PURPOSE: Cockroach (CR) is a common source of indoor allergens, and Per a 1 is a major American CR (Periplaneta americana) allergen; however, several attributes of this protein remain unknown. This study identifies a novel specific B cell epitope and anatomical locations of Per a 1.0105. METHODS: Recombinant Per a 1.0105 (rPer a 1.0105) was used as BALB/c mouse immunogen for the production of monoclonal antibodies (MAb). The MAb specific B cell epitope was identified by determining phage mimotopic peptides and pair-wise alignment of the peptides with the rPer a 1.0105 amino acid sequence. Locations of the Per a 1.0105 in P. americana were investigated by immunohistochemical staining. RESULTS: The rPer a 1.0105 (~13 kDa) had 100%, 98% and > or =90% identity to Per a 1.0105, Per a 1.0101, and Cr-PII, respectively. The B-cell epitope of the Per a 1.0105 specific-MAb was located at residues99 QDLLLQLRDKGV110 contained in all 5 Per a 1.01 isoforms and Per a 1.02. The epitope was analogous to the Bla g 1.02 epitope; however, this B-cell epitope was not an IgE inducer. Per a 1.0105 was found in the midgut and intestinal content of American CR but not in the other organs. The amount of the Per a 1 was ~544 degrees Cg per gram of feces. CONCLUSIONS: The novel Per a 1 B-cell epitope described in this study is a useful target for allergen quantification in samples; however, the specific MAb can be used as an allergen detection reagent. The MAb based-affinity resin can be made for allergen purification, and the so-purified protein can serve as a standard and diagnostic allergen as well as a therapeutic vaccine component. The finding that the Per a 1 is contained in the midgut and feces is useful to increase yield and purity when preparing this allergen.

Preparation and identification of antibodies against ADMA / 国际检验医学杂志

Objective To prepare and identify the monoclonal antibody against asymmetric dimethylarginine (ADMA) and to do further research on clinical application .Methods The short immune peptide antigen was synthetized by solid phase technology . Splenocytes from the Balb /c mice immunized by synthetized antigen were fused with myeloma cells SP 2/0 for producing hybrido-ma .Hybridoma cell line secreting antibodies against ADMA was sifted by indirect ELISA and limiting dilution assay .The specificity of monoclonal antibodies against human ADMA were evaluated with Western blot and ELISA .Results Two hybridoma cell lines stably secreting monoclonal antibodies against ADMA were developed and named 22-1-1and 38-1-6 respectively .By applying West-ern blot and ELISA ,the results indicated that all monoclonal antibodies raised could specifically react with ADMA .Conclusion The success in the production of ADMA monoclonal antibody establishes foundation for application in the practice of clinical labora-to ry .

Preparation of Monoclonal Antibody of Sodium Estrone Sulfate from Pregnant Mare's Urine / 医药导报

Objective To establish a method to prepare anti-sodium estrone sulfate monoclonal antibody ( ESS-Mab) . Methods Balb/c mice were immunized by ESS. Immune methods were screened. The blood serum potencies were measured by indirect ELISA and the best consistence of antigen and the first antibody were confirmed with method of titration. Cell fusion was carried by using PEG method and McAb hybridoma was screened with the indirect ELISA. Results The best immunization method of mice was subcutaneously multi-point injection in mouse back with the dose of 200/100 μg ESS antigen five times. The fusion rate was 90. 2%. Hybridoma positive rate of ELISA screening was 4. 4%. Finally two cell lines 2C8 and 8A7 with good specificity and sensitivity were obtained. Conclusion The best immunization way is selected and indirect ELISA is set up effectively and reliably for screening and presenting ESS McAb. the hybridoma technique is able to prepare monoclonal antibody of anti-ESS successfully.

Drugs obtained by biotechnology processing / Drogas obtidas pelo processamento biotecnológico

In recent years, the number of drugs of biotechnological origin available for many different diseases has increased exponentially, including different types of cancer, diabetes mellitus, infectious diseases (e.g. AIDS Virus / HIV) as well as cardiovascular, neurological, respiratory, and autoimmune diseases, among others. The pharmaceutical industry has used different technologies to obtain new and promising active ingredients, as exemplified by the fermentation technique, recombinant DNA technique and the hybridoma technique. The expiry of the patents of the first drugs of biotechnological origin and the consequent emergence of biosimilar products, have posed various questions to health authorities worldwide regarding the definition, framework, and requirements for authorization to market such products.

Nos últimos anos, tem aumentado exponencialmente o número de fármacos de origem biotecnológica ao dispor das mais diversas patologias, entre elas destacam-se, os diferentes tipos de cancêr, as doenças infecciosas (ex. vírus AIDS/HIV), as doenças autoimunes, as doenças cardiovasculares, a Diabetes Mellitus, as doenças neurológicas, as doenças respiratórias, entre outras. A indústria farmacêutica tem recorrido a diferentes tecnologias para a obtenção de novos e promissores princípios ativos, como são exemplo a fermentação, a técnica de DNA Recombinante, a técnica de hidridoma, entre outras. A queda das patentes dos primeiros fármacos de origem biotecnológica e o consequente aparecimento dos produtos biossimilares têm colocado diferentes questões às autoridades de saúde mundiais, sobre a definição, enquadramento e exigências para a autorização de entrada no mercado deste tipo de produtos.

Quantification of Der f 1 in houses of patients allergic to house dust mite, Dermatophagoides farinae, using a locally produced detection reagents.

Background: House dust mite (HDM) allergen quantification in house dust samples before and after the allergen elimination is one means of convincing the target population about the health benefits of allergen removal from their environment. Objective: To produce local reagents for quantification of Der f 1 (major allergen of Dermatophagoides farinae) in dust samples from houses of HDM allergic Thai patients. Methods: Recombinant Der f 1 was used for immunization of a BALB/c mouse for hybridoma production. Polyclonal antibody (PAb) to whole body extract of D. farinae was prepared from an immunized rabbit. A sandwich ELISA (MAb-allergen-PAb) was used, in comparison with the commercialized reagents (Indoor Biotechnology, UK), to quantify Der f 1 in dust samples. Results: Two hybridoma clones, Df1-1 and Df1-2, were established. Their secreted MAbs (MAbDf1-1 and MAbDf1-2, respectively) bound to the homologous antigen as well as native Der f 1 and a crude extract of D. farinae. Epitopes of MAbDf1-1 and MAbDf1-2 were located at amino acid residues 206NSQHYGISNYCQ217 and 283DYW---NSWD-WGDSG298 of Der f 1. MAbDf1-1 had higher affinity to Der f 1 than the MAbDf1-2. A sandwich ELISA (MAbDf1-1-allergen-PAb) and commercialized reagents (MAb1-allergen-MAb2 sandwich ELISA) were used in comparison for quantification of Der f 1 in 42 dust samples collected from bedrooms and living rooms of 21 houses of the HDM allergic patients. All of the 42 dust samples measured by both ELISAs had the Der f 1 levels higher than 2 mg per gram of fine dust which is the HDM allergy sensitizing level. In addition, Der f 1 levels in 41 samples (except 1 sample from a living room) measured by the MAbDf1-1-PAb and MAb1-MAb2 sandwich ELISAs were higher than 10 mg per g of dust which is the morbidity level of HDM allergen. The local sandwich ELISA showed a high coefficient correlation (r = 0.91) in measuring known amounts of recombinant and native Der f 1. The results indicate that the reagents produced in the present study can be used for measuring the environmental levels of HDM Der f 1. The assay can also be used for standardization of the HDM extract for monitoring patient's allergenic status or for immunotherapeutic purpose.

Preparation and characterization of two monoclonal antibodies against human VSIG4 / 中国免疫学杂志

Objective: To prepare anti-VSIG4 monoclonal antibodies and characterize their biological functions.Methods: BALB/c mice were immunized with transfected cell line (L929/VSIG4L) as immunogen.The spleen B cells of the mice were fused with SP2/0 and hybridoma cells were screened with transfected cell line (L929/VSIG4) by FCM.After acquisition of the hybridomas secreting anti-VSIG4 mAb,their biological activities were investigated by indirect immunofluorescence,Western blot,competitive inhibition test,and MTT assay.Results:Two stable hybridomas,9A7 and 9D5 were obtained,which could continuously secrete specific anti-VSIG4 monoclonal antibodies.The following biological activity studies showed that these monoclonal antibodies could recognize the natural VSIG4 expressed on the macrophages and several cancer cell lines,such as Jurkat,THP-1 and H446.Furthermore,they could block the inhibitory effects of VSIG4 on proliferation of T cells in vitro.Conclusion: Two hybridomas secreting anti-VSIG4 monoclonal antibodies have been established.These monoclonal antibodies provide useful tools for further studying VSIG4's biological function and its unknown receptor.

Preparation and identification of anti-HIV-1 gp120 monoclonal antibody / 中国生化药物杂志

Purpose To Prepare anti- HIV-1 gp120 monoclonal antibodies and to identify the specificity of antibodies in order to provide technique for preparing HIV remedial antibodies. Methods The gene fragment of HIV-1 gp120 was connected to PEGX-4T-2 prokaryotic expressing vector. The vector was cut by enzyme. GST-HIV protein was expressed by E. coli XL1-blue. The BALB/c mice were immunized with purified GST-HIV antigen, and then the fusion of mice spleen cell and myeloma cell SP2/0 was executed as the routine cell-fusion technique. Positive cells were screened by Indirect ELISA. Immuno-blotting assay and Western blot identified the specificity of antibodies. Results External gene section from the recombinant plasmid by sequencing showed the same size of HIV-1 gp120 gene sequences. An external expressed protein band of 32 KD was obtained after purified protein SDS-PAGE electrophoresis. It indicated that six strains of hybridoma cells secreting special monoclone antibodies had been obtained. ELISA results showed that six strains monoclonal antibodies only reacted to HIV-1 gp120 antigen. Western blot results showed that a band with molecular weight 32 kDa was obtained, which could interact with HIV-1 gp120 monoclonal antibody. Conclusion Six strains of hybridoma cells secreting special monoclone antibodies had been obtained. The prepared monoclonal antibodies have established a basis for HIV remedial antibody.

Preparation and purification of monoclonal antibodies against human chorionic gonadotrophin / 中国生化药物杂志

Purpose To prepare monoclonal antibody against hCG. Methods Balb/c mice were immunized with hCG, and spleen cells from the mice were fused with myeloma cells SP2/0 at ratio of 5:1. Positive clones were screened by indirect enzyme-linked-immunoadsorbent assay (ELISA) ,then the clones were subcloned by limiting dilution and amplified further. After intraperitoneal injection of hybridoma cells, mouse ascites antibody was prepared. Titres and specificity of the ascites antibody were identified and determined by indirect ELISA. The ascites were purified by protein A affinity chromatography. Results Two strains of hybridoma cell lines obtained could secrete MAb stably. The titre of MAb of cell culture supernate is more than 10~(-3), and the titre of prepared ascites MAb is more than 10~(-7).The purity of the obtained monoclonal antibody was more than 98% and recovery reached 75 % after purification. Conclusion The obtained two strains of hybridoma cell lines can secrete MAb stably. The monoclonal antibodies can be used in the research of early pregnancy and tumor diagnosis .

Production of Monoclonal Antibodies Specific to FimA of Porphyromonas gingivalis and Their Inhibitory Activity on Bacterial Binding

BACKGROUND: The FimA of Porphyromonas gingivalis is a crucial pathogenic component of the bacteria and has been implicated as a target for vaccine development against the periodontal diseases. METHODS: In this study, the purified fimbriae (FimA subunit polymers) protein was used for immunization in their native form and B hybridoma clones producing antibodies specific to FimA were established. RESULTS: The monoclonal antibodies prepared from selected two clones, designated #123 (IgG2b/ kappa) and #265 (IgG1/kappa), displayed different patterns of binding activity against the cognate antigen. Both antibodies reacted with conformational epitopes expressed by partially dissociated oligomers, but not with monomer as elucidated by Western blot analysis. Ascites fluid containing the monoclonal antibodies showed the inhibitory activity against P. gingivalis to saliva-coated hydroxyapatite beads, an in vitro model for the pellicle-coated tooth surface. CONCLUSION: These results suggest that the monoclonal antibodies could be used as vaccine material against the periodontal diseases through passive immunization.

Preparation and characterization of a mouse anti-human CD40 mutant monoclonal antibody / 细胞与分子免疫学杂志

AIM: To prepare and characterize a mouse anti-human CD40 mutant monoclonal mAb. METHODS: Female BALB/c mice of 6-8 weeks old were immunized with CD40 mutant transfectant (L929-CD40mu) as immunogen. The spleen B cells of the mice were fused with Sp2/0 myeloma cells. The hybridoma cells were screened with CD40 mutant transfectant (L929-CD40mu) by FCM. Faststrip analysis was performed to identify Ig subclass of this mAb. The epitope recognized by this mAb was detected by Bio-5C11 competitive assay. Western blot technique was adopted to identify the mAb. The proliferation of tumor cells in vitro was analyzed by MTT assay and apoptosis of tumor cells in vitro was analyzed by PI-annexin V assay. RESULTS: One hybridoma cell line named 10C5 was obtained, which had the property of secreting anti-human CD40 mutant monoclonal antibody continuously and steadily. This mAb specifically recognized human CD40 mutant molecule and induced the apoptosis of tumor cells in vitro. CONCLUSION: One hybridoma cell line which can secret a mouse anti-human CD40 mutant mAb has been prepared successfully. This mAb can inhibit the growth of tumor cells expressing CD40 mutant and induce their apoptosis in vitro.