A monoclonal antibody-based icELISA for puerarin / 中国中药杂志

Puerarin was conjugated with bovine serum albumin(BSA) and ovalbumin(OVA) by periodate oxidation to serve as the immunogen and coating antigen, respectively. BALB/c mice were immunized with puerarin-BSA according to the routine immunization procedure, and the titer and specificity of serum were detected after three immunization. After booster immunization, mouse spleen lymphocytes were fused with mouse myeloma cells, and 24 hybridoma cell lines of the monoclonal antibodies against puerarin were screened by monoclonal antibody screening technique. Ascites was prepared and purified. The cross-reactivity of monoclonal antibody(mAb) M1 with 4'-methoxy puerarin, daidzin, puerarin-6″-O-xyloside, daidzein, mirificin, 3'-methoxy puerarin, and 3'-hydroxy puerarin was 239.84%, 112.18%, 67.89%, 58.28%, 22.37%, 0.40%, and 0.20%, respectively, and those with other analogs such as baicalein and baicalin were all less than 0.10%. The IC_(50) and the working range of the indirect competitive enzyme-linked immunosorbent assay(icELISA) for puerarin were 44.80 ng·mL~(-1) and 8.20-292.30 ng·mL~(-1), respectively. The average recovery was 91.95%-98.20% with an RSD in the range of 0.70%-2.60%. The content of puerarin in different Puerariae Lobatae Radix samples was determined with icELISA and validated by UPLC-MS. The correlation between data obtained from icELISA and UPLC-MS was 0.999 0, indicating that icELISA is suitable for the rapid detection of puerarin in Puerariae Lobatae Radix samples.

Production and Characterization of Anti-Staphylococcal Toxic Shock Syndrome Toxin-1 Monoclonal Antibody / 대한진단검사의학회지

BACKGROUND: Recently the association between the virulence factors of Staphylococcus aureus and the outcome of the patients infected with the organism appears to be the subject of active investigation. Toxic shock syndrome toxin-1 (TSST-1) is thought to be a clinically more significant virulence factor than other staphylococcal toxins. We attempted to produce and characterize monoclonal antibodies to staphylococcal TSST-1. METHODS: An important epitope of TSST-1, amino acids 1-15 region, was synthesized into a peptide antigen, and Balb/c mice were immunized by intraperitoneal injection of the synthetic antigen. Hybridomas were produced by fusing immunized murine splenocytes with immortal myeloma cells. Hybridomas were cloned through a limiting dilution method. Stable cultured hybridoma was injected into the peritoneal cavity of Balb/c mice, and peritoneal fluid containing the monoclonal antibody was produced. RESULTS: One IgG2b type monoclonal antibody and two IgM type monoclonal antibodies were obtained. The IgG2b type monoclonal antibody was able to detect 5 microgram of TSST-1 with Western blot analysis and showed a strong reactivity to TSST-1 with ELISA. CONCLUSIONS: Highly immunoreactive anti-TSST-1 monoclonal antibody was produced by the use of synthesized peptide antigen. Diagnostic and protective capacity of this monoclonal antibody should be evaluated in the future.

The non-palindromic adaptor-PCR method for the identification of the T-cell receptor genes of an interferon-gamma-secreting T-cell hybridomaspecific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen

Cloning of the T-cell receptor genes is a critical step when generating T-cell receptor transgenic mice. Because T-cell receptor molecules are clonotypical, isolation of their genes requires reverse transcriptase-assisted PCR using primers specific for each different Valpha or Vß genes or by the screening of cDNA libraries generated from RNA obtained from each individual T-cell clone. Although feasible, these approaches are laborious and costly. The aim of the present study was to test the application of the non-palindromic adaptor-PCR method as an alternative to isolate the genes encoding the T-cell receptor of an antigen-specific T-cell hybridoma. For this purpose, we established hybridomas specific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen. These T-cell hybridomas were characterized with regard to their ability to secrete interferon-gamma, IL-4, and IL-10 after stimulation with the antigen. A CD3+, CD4+, CD8- interferon-gamma-producing hybridoma was selected for the identification of the variable regions of the T-cell receptor by the non-palindromic adaptor-PCR method. Using this methodology, we were able to rapidly and efficiently determine the variable regions of both T-cell receptor chains. The results obtained by the non-palindromic adaptor-PCR method were confirmed by the isolation and sequencing of the complete cDNA genes and by the recognition with a specific antibody against the T-cell receptor variable ß chain. We conclude that the non-palindromic adaptor-PCR method can be a valuable tool for the identification of the T-cell receptor transcripts of T-cell hybridomas and may facilitate the generation of T-cell receptor transgenic mice.

Getting higher yields of monoclonal antibody in culture.

Getting higher yields of monoclonal antibody (MAb) is a problem in Hybridoma Technology which has two major bottlenecks: a) poor yield of hybridized cells, b) low cellular productivity of MAb in culture. There are three ways of obtaining high MAb yield in vitro a) Large scale culture, b) high density culture and c) enhancing individual cellular productivity in culture. Currently, the focus is on the correct synergistic combination of fortified nutrient media, bioreactor design and mode of operation. Maximization of cell culture longevity, maintenance of high specific antibody secretion rates, nutrient supplementation, waste product minimization and control of environmental conditions are important parameters for improvement of large scale production of MAb. Though, MAb yields have improved rapidly over the decade, there is a growing concern for the decrease in quality of MAb secreted. Further research is therefore necessary to take full advantage of MAb as a potential diagnostic agent for in vivo therapy.

production of murine monoclonal antibodies [MAb] directed against human T-lymphocyte subsets

The production of murine monoclonal antibody [MAb] has not yet been reported in Iran. The present work describes for the first time the generation of several murine hybridoma clones secreting MAbs directed against human leukocyte surface antigens. The secreted antibodies by hybridoma clones have been screened on different lymphoid and non-lymphoid tissues. Results indicated that of seven hybridomas, clones 3F11, 3C3 and 1F2 showed a strong reactivity for T-cells purified from thymus and tonsil tissues. Moreover, clones 1 D4 and 6G5 which partially stained thymus tissues were found to be negative on purified B-cells and monocytic cell line U937. The third group, 4E5 and 4F4 hybridoma clones was expressed weakly on purified T, B-cells and U937 cell line. Further work is needed to determine the epitopes recognized by these MAbs and a comparison of the data with those presented at the previous leukocyte antigens workshop

Generación de hibridomas humano-humano secretores de anticuerpos empleando la línea celular linfoblastoide W1-L2-729 HF2 / Genaration of antibody secretor human-human hybridomas using W1-L2-729 HF2 lymphoblastic cell line

En el presente trabajo se informa por primera vez en Cuba, la producción de hibridomas humano-humano secretores de inmunoglobulinas a partir de la fusión de linfocitos de ganglios linfáticos axilares de paciente con cámcer de mama con células de línea celular linfoblastoide B humana W1-L2-729HF2. En 3 de las 4 fusiones realizadas se obtuvieron hibridomas con crecimiento celular en 196 de los 569 pozos sembrados con el producto de las fusiones. Los primeros híbridos se observaron al final de la primera semana, aunque la mayoría se detectó en la segunda y tercera semanas de haberse realizado la fusión. De los 196 pozos con crecimiento celular, 120 eran secretores de inmunoglobulinas. El nivel de secreción de los híbridos clonados fue desde 4,5 hasta 8,0 *g/mL por 10 6 células. La naturaleza híbrida se determinó por análisis cromosómico y por la clase de inmunoglobulina producida. No se detectaron anticuerpos específicos contra los antígenos intracelulares o localizado en la superficie celular de las diferentes líneas celulares tumorales humanas empleadas en el estudio

Anticuerpos monoclonales en urología / Monoclonal antibodies in urology

En este artículo hace una revisión de los aspectos más relevantes y actuales acerca de los anticuerpos monoclonales. Se realiza una breve revisión histórica y de evolución a través de los últimos años sobre la fabricación y la obtención de los anticuerpos monoclonales a partir de hibridomas, mencionando los pasos básicos requeridos para lograr la fusión celular y la selección de los anticuerpos deseados. Se hace hincapié en cuanto al beneficio que representan para el urólogo y los usos clínicos en cuanto a diagnósticos y tratamiento en carcinomas de próstata y cáncer de urotelio y, así mismo, para el de los pacientes con transplante renal. Por último, se hacen algunas conclusiones en cuanto al presente y el futuro que los anticuerpos monoclonales tendrán en urología

Anticuerpos monoclonales contra el antígeno carcinoembrionario en ensayos inmunohistoquímico e inmunoquimicos / Monoclonal antibodies against carcinoembryonic antigen in immunohistochemical and immunochemical assays

En el presente trabajo se reporta la obtención de cinco clones de hibridomas de ratón, secretores de anticuerpos monoclonales que reconocen una preparación de antígeno carcinoembrionario (CEA). Durante el proceso de tamizaje y selección de estos clones, se empleó fundamentalmente un sistema de radioinmunoensayo, donde se compararon los sobrenadantes de cultivo o los ascitis tumorales con un antisuero policlonal de referencia, con respecto a su enlazamiento y especificidad por 125I-CEA. Los anticuerpos monoclonales seleccionados pertenecen a las clases G y M de inmunoglobulinas de ratón, y dos de ellos manifestaron bajo o ningún reconocimiento del antígeno de reactividad cruzada no específico (NCA). Los anticuerpos IOR-CEA 3 (IgM) e IOR-CEA 5 (IgG1) se estudiaron para su reconocimiento de tejidos fetales humanos, lesiones benignas de la mama, y cánceres de mama, pulmón y colon. El IOR-CEA 3 puede ser utilizado para estudios de confirmación histológica diagnóstica por técnica de inmunofluorescencia indirecta en tejido fresco. Los anticuerpos IOR-CEA 1 (IgG1) e IOR-CEA 2 (IgM) se ensayaron mediante diferentes combinaciones en un sistema ELISA para conocer su capacidad de detectar CEA purificado. Los resultados preliminares indican un adecuado nivel de reconocimiento cuando se utiliza como primer anticuerpo el antisuero policlonal anti-CEA de referencia y el monoclonal IOR-CEA 1 como segundo anticuerpo

Producción de un anticuerpo monoclonal para la detección de antígeno A del sistema ABO de grupos sanguíneos / Production of a monoclonal antibodies for the detection of antibody A of the ABO blood group system

Se demuestra en el presente trabajo la utilidad de anticuerpos monoclonales anti-grupo sanguíneo. A obtenidos a partir de un híbrido generado por fusión entre linfocitos de bazo de ratón inmunizado y células de mieloma. El clon productor del anticuerpo monoclonal, denominado B2/C114, fue propagado como tumor ascítico en ratones, lo que permitió su conservación y producción en gran escala. El reactivo obtenido es útil para la evaluación rutinaria de grupos sanguíneos, presentando gran avidez, estabilidad superior a 12 meses y alto título, condiciones que satisfacen ampliamente los requerimientos de seguridad diagnóstica y costo efectivo