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Antibody-Based Therapies
Published in David E. Thurston, Ilona Pysz, Chemistry and Pharmacology of Anticancer Drugs, 2021
Laboratory animals (usually mice) are first exposed to the antigen of interest by a series of injections over the course of several weeks, sometimes supplemented by in vivo electroporation of the antigen which significantly enhances the immune response. Splenocytes (i.e., B cells) are then isolated from the spleen, and these are fused with immortalized myeloma cells using electrofusion, which causes the B cells and myeloma cells to align and fuse under the influence of an electric field. The myeloma cells are selected beforehand to ensure they are not secreting antibody themselves, and that they lack the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) gene which causes them to be sensitive to the HAT medium.
Prolactin Receptors in Normal Tissues and in Animal Models for Breast Cancer
Published in Nagasawa Hiroshi, Prolactin and Lesions in Breast, Uterus, and Prostate, 2020
Paul A. Kelly, David Gould, Hiroaki Okamura, Jean Dijane
BALB/c mice (female) were injected with 9 to 10 μg of partially purified receptors (binding capacity 150 pmol/mg protein four times over a period of 9 weeks as follows: 1st injection, intraperitoneal with an equal volume of Freund’s complete adjuvant; 2nd (at 3 weeks) and 3rd (at 6 weeks), intraperitoneal with an equal volume of Freund’s incomplete adjuvant; and 4th (at 9 weeks), subcutaneous or intravenous injection with an equal volume of saline. Three days after the final injection, spleen cells were removed and fused with SP2/0 myeloma cells at a ratio of 10:1 (spleen cell:myeloma) using polyethylene glycol 1000. Cell suspensions were diluted with RPMI 1640 medium containing 10% fetal calf serum and plated into 96-well microtiter plates. The next day, O.lmf of hypoxanthine/aminopterin/thymidine (HAT) medium was added to each well and hybridoma cells were selected.
Cellular and Viral Oncogenes
Published in Pimentel Enrique, Oncogenes, 2020
The foci of transformed cells can be picked up and single cells from them can be cloned in semisolid media. Clonal cells can be grown to mass culture and their DNAs can be analyzed for detection of repetitive sequences, for example, human Alu sequences.120,121 The putative oncogenes present in donor DNA can be studied by transformation assays of DNA fragments obtained by digestion with restriction enzymes.118 Since the transforming ability of extracted DNA is somewhat variable, it is convenient to monitor the donor DNA with some well characterized marker in parallel assays. The most used of these markers is the thymidine kinase (tk) gene, which is assayed in murine LMtk- cells, the tk+ colonies being selected in HAT medium.122
Current understanding of Lesch-Nyhan disease and potential therapeutic targets
Published in Expert Opinion on Orphan Drugs, 2019
Experimental and clinical data suggest that the transfer of the HPRT1 gene into brain cells appears to be necessary to correct the neurological dysfunction of this disorder, and, possibly, at early stages of neuronal development. Initial studies demonstrate the transfer and transcription of HPRT gene in mice and higher-order mammals brain cells, by direct in vivo infection with recombinant adenovirus or herpes simplex virus type 1 vectors [105–107]. In 1962, Szybalski et al. [109] isolated DNA form HPRT-positive cells and used it to transform HPRT-negative cells. The transformed cells did not die in the presence of the HAT medium (in which only cells with HPRT activity could survive). However, although this study in HPRT1 gene became the first documented evidence of heritable gene transfer in mammalian cells, since 1996 no research on HPRT1 gene transfer has been reported. Successful recent reports of brain enzyme delivery or brain gene therapy with adenoviral vectors offer hope for the treatment of other metabolic neurological diseases, such as LND.
Determination of deoxynivalenol by ELISA and immunochromatographic strip assay based on monoclonal antibodies
Published in Toxin Reviews, 2021
Tingting Yan, Qi Zhang, Du Wang, Peiwu Li, Xiaoqian Tang, Wen Zhang
One week after the last immunization, serum was collected from the tail vein of each mouse, and an ic-ELISA was used to determine the antiserum titer. Three days before cell fusion, the mouse that showed the highest antibody titer and sensitivity was given an additional intraperitoneal injection of 100 μg DON-BSA in PBS without adjuvant. The spleen of the immunized mice and myeloma cells Sp2/0 were aseptically isolated, and fused at a ratio of 10:1 in the presence of PEG1500. Hybridoma cells were cultured in 96-well plates with HAT medium and incubated at 37 °C and 5% CO2. Ic-ELISA was used to screen the anti-DON positive clones and DON was used as the competitive inhibitor. After cell culture, a hybridoma cell was screened.