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A novel method that optimizes the screening for antibody-secreting hapten-specific hybridoma cells by using flow cytometry is described. Cell clones specific for five different haptens were analyzed. We selectively double stained and analyzed fixed hybridoma cells with fluorophore-labeled haptens to demonstrate the target-selectivity, and with a fluorophore-labeled anti-mouse IgG antibody to characterize the level of surface expression of membrane-bound IgGs. ELISA measurements with the supernatants of the individual hybridoma clones revealed that antibodies from those cells, which showed the highest fluorescence intensities in the flow cytometric analysis, also displayed the highest affinities for the target antigens. The fluorescence intensity of antibody-producing cells corresponded well with the produced antibodies' affinities toward their respective antigens. Immunohistochemical staining verified the successful double labeling of the cells. Our method makes it possible to perform a high-throughput screening for hybridoma cells, which have both an adequate IgG production rate and a high target affinity. (C) 2014 Elsevier B.V. All rights reserved.
The large scale production of a monoclonal anti-progesterone antibody in serum free medium followed by affinity chromatography on protein G lead to a contamination of the antibody sample with a protein of about 14 kDa. This protein was identified by mass spectrometry as secretory leukocyte protease inhibitor (SLPI). This SLPI contamination lead to a failure of the fiber-optic based competitive fluorescence assay to detect progesterone in milk. Purification of the monoclonal antibody using protein A columns circumvented this problem.
Tumor antigen-specific T cell clones represent a useful tool in tumor immunology; however, their long-term culture is limited. To generate an immortalized cytotoxic T cell clone against the human tumor antigen mucin, we exposed a previously generated T cell culture to Herpesvirus saimiri. We obtained an immortalized human CD4+ T cell clone, termed SITAM. Clonality of these cells was shown by analysis of the alpha/beta-T cell receptor (TCR) repertoire. Cytolytic activity was demonstrated against several mucin-expressing tumor cell lines and could not be detected against non-mucin-expressing cells. SITAM cells maintained their features stably for 2 years. Furthermore, growth of the tumor cell line Capan-2 in NOD/SCID mice was inhibited when SITAM cells were coinjected subcutaneously with tumor cells. SITAM cells provide an unlimited source of clonal T cells for analysis of tumor recognition and may be of help in TCR-targeted immunotherapy.
BACKGROUND: Dendritic cells (DC) as antigen presenting cells play an important role in immunotherapy of cancer. Mucin, encoded by the gene MUC1, is a human tumor antigen expressed in breast, pancreatic and ovarian cancers. Therefore, MUC1-transfected DC would be an attractive tool in constructing cancer vaccines. MATERIALS AND METHODS: Using two different cationic liposome preparations and, for comparison, a recombinant adenovirus expressing mucin, we tested the efficiency of mucin gene transfer into DC by flow cytometry. We investigated if these transfected DC were able to specifically stimulate autologous peripheral blood lymphocytes (PBL) from healthy donors. RESULTS: Flow cytometry revealed that 5-20% of DC transfected with liposomes Lipofectin and 20-40% of DC transduced with adenovirus expressed the relevant mucin epitopes. The expression of mucin on DC was similar to the expression of mucin found on carcinoma cells. After antigen uptake, DC specifically stimulated autologous PBL. CONCLUSION: We have shown that cationic liposomal gene transfer into human DC was feasible. We could obtain antigen specific stimulation of PBL at a similar rate as with adenoviral MUC1-transduced DC.
Dendritic cells (DCs) are the most potent antigen-presenting cells of the immune system and are currently being investigated in clinical applications as cancer vaccines. An efficient cryopreservation method would greatly contribute to their use in clinical trials. We have established a method for freezing of DCs derived from peripheral blood mononuclear cells using the plasma expander Gelifundol. This enabled us to reduce the concentration of the toxic DMSO to 5%. The method could be performed without the addition of fetal calf serum or any other serum. After freezing, the viability of the DCs was 90%. The cells exhibited all the phenotypic characteristics (CD11c+, HLA-DR+, CD80+, CD83+, CD86+) of DCs, as tested by flow cytometry. Cells transfected with cDNA for the tumour antigen mucin expressed this protein on their surfaces in the same manner as before freezing. The stimulating capacity of a mixed lymphocyte culture was also preserved. These findings offer an efficient method for the cryopreservation of DCs for use in clinical trials.
A competitive immunoassay to detect a hapten using an enzyme-labelled peptide mimotope as tracer
(2002)
Mimotope peptides-peptides which mimic the binding of a hapten to its corresponding monoclonal antibody-were conjugated to peroxidase and used in competitive immunoassay. The established immunoassay was used to quantitatively determine the concentration of hapten. As model system in all the experiments described here, we used the binding of the monoclonal antibody B13-DE1 to fluorescein and the corresponding peptide mimotope.