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Cloning and characterization of a single chain antibody to glucose oxidase from a murine hybridoma
(2007)
Glucose oxidase (GOD) is an oxidoreductase catalyzing the reaction of glucose and oxygen to peroxide and gluconolacton (EC 1.1.3.4.). GOD is a widely used enzyme in biotechnology. Therefore the production of monoclonal antibodies and antibody fragments to GOD are of interest in bioanalytics and even tumor therapy. We describe here the generation of a panel of monoclonal antibodies to native and heat inactivated GOD. One of the hybridomas, E13BC8, was used for cloning of a single chain antibody (scFv). This scFv was expressed in Escherichia coli XL1-blue with the help of the vector system pOPE101. The scFv was isolated from the periplasmic fraction and detected by western blotting. It reacts specifically with soluble active GOD but does not recognize denatured GOD adsorbed to the solid phase. The same binding properties were also found for the monoclonal antibody E13BC8.
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 mussel Mytilus edulis can be used as model to study the molecular basis of reproductive isolation because this species maintains its species integrity, despite of hybridizing in zones of contact with the closely related species M. trossulus or M. galloprovincialis. This study uses selective antibody production by means of hybridoma technology to identify molecules which are involved in sperm function of M. edulis. Fragmented sperm were injected into mice and 25 hybridoma cell clones were established to obtain monoclonal antibodies (mAb). Five clones were identified producing mAb targeting molecules putatively involved in sperm function based on enzyme immunoassays, dot and Western blotting as well as immunostaining of tissue sections. Specific localization of these mAb targets on sperm and partly also in somatic tissue suggests that all five antibodies bind to different molecules. The targets of the mAb obtained from clone G26-AG8 were identified using mass spectrometry (nano-LC-ESI-MS/MS) as M6 and M7 lysin. These acrosomal proteins have egg vitelline lyses function and are highly similar (76%) which explains the cross reactivity of mAb G26- AG8. Furthermore, M7 lysin was recently shown to be under strong positive selection suggesting a role in interspecific reproductive isolation. This study shows that M6 and M7 lysin are not only found in the sperm acrosome but also in male somatic tissue of the mantle and the posterior adductor muscle, while being completely absent in females. The monoclonal antibody G26-AG8 described here will allow elucidating M7/M6 lysin function in somatic and gonad tissue of adult and developing animals.
Immunochemical analytical methods are very successful in clinical diagnostics and are nowadays also emerging in the control of food as well as monitoring of environmental issues. Among the different immunoassays, luminescence based formats are characterized by their outstanding sensitivity making this format especially attractive for future applications. The need for multiparameter detection capabilities calls for a tool box of dye labels in order to transduce the biochemical reaction into an optically detectable signal. Here, in a multiparameter approach each analyte may be detected by a different dye with a unique emission color (covering the blue to red spectral range) or a unique luminescence decay kinetics. In the case of a competitive immunoassay format for each of the different dye labels an individual antibody would be needed. In the present paper a slightly modified approach is presented using a 7-aminocoumarin unit as the basic antigen against which highly specific antibodies were generated. Leaving the epitope region in the dyes unchanged but introducing a side group in positon 3 of the coumarin system allowed us to tune the optical properties of the coumarin dyes without the necessity of new antibody generation. Upon modification of the parent coumarin unit the full spectral range from blue to deep red was accessed. In the manuscript the photophysical characterization of the coumarin derivatives and their corresponding immunocomplexes with two highly specific antibodies is presented. The coumarin dyes and their immunocomplexes were characterized by steady-state and time-resolved absorption as well as emission spectroscopy. Moreover, fluorescence depolarization measurements were carried out to complement the data stressing the different binding modes of the two antibodies. The binding modes were evaluated using the photophysics of 7-aminocoumarins and how it was affected in the respective immunocomplexes, namely, the formation of the intramolecular charge transfer (ICT) as well as the twisted intramolecular charge transfer (TICT). In contrast to other antibody-dye pairs reported a distinct fluorescence enhancement upon formation of the antibody-dye complex up to a factor of SO was found. Because of the easy emission color tuning by tailoring the coumarin substitution for the antigen binding in nonrelevant position 3 of the parent molecule, a dye tool box is on hand which can be used in the construction of competitive multiparameter fluorescence enhancement immunoassays (FenIA).
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 recombinant single chain antibody fragment (designated scDE1) of the murine monoclonal anti-fluorescein antibody B13-DE1 was generated using the original hybridoma cells as source for the variable antibody heavy and light chain (VH and VL) genes. After cloning the variable genes into a phage vector a functional antibody fragment was selected by phage display panning. Recombinant antibody could be expressed as phage antibody and as soluble single chain antibody in Escherichia coli. High yield of scDE1 could also be detected in bacterial culture supernatant. The scDE1 showed the same binding specificity as the parental monoclonal antibody, i.e. it bound fluorescein, fluorescein derivatives and a fluorescein peptide mimotope. Surface plasmon resonance revealed a K(D) of 19 nM for the scDE1 compared to 0.7 nM for the monoclonal antibody. The isolated soluble scDE1 could easily be conjugated to horseradish peroxidase which allowed the use of the conjugate as universal indicator for the detection of fluorescein-labelled proteins in different immunoassays. Detection of hCG in urine was performed as a model system using scDE1. In addition to E. coli the scFv genes could also be transferred and expressed in eukaryotic cells. Finally, we generated HEK293 cells expressing the scDE1 at the cell surface.
A monoclonal antibody against the potential tumor suppressor kinase-enhanced protein phosphatase 1 (PP1) inhibitor KEPI (PPP1R14C) was generated and characterized. Human KEPI was expressed in Escherichia coli and used to immunize Balb/c mice. Using hybridoma technology, one clone, G18AF8, was isolated producing antibodies which bound specifically to the KEPI protein in ELISA, immunoblotting and flow cytometry. The antibody was also successfully applied to stain KEPI protein in paraffin sections of human brain. The epitope was mapped using peptide array technology and confirmed as GARVFFQSPR. This corresponds to the N-terminal region of KEPI. Amino acid substitution analysis revealed that two residues, F and Q, are essential for binding. Affinity of binding was determined by competitive ELISA as 1 mu M. In Western blot assays testing G18AF8 antibody on brain samples of several species, reactivity with hamster, rat and chicken samples was found, suggesting a broad homology of this KEPI epitope in vertebrates. This antibody could be used in expression studies at the protein level e.g. in tumor tissues.
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.
The influence of interleukin 4 (IL-4) on antibody titer in serum and spleen culture supernatant in mice immunized with spinach (Spinacia oleracea L.) Rubisco was investigated. Therefore, we boosted one mouse additionally to the antigen with recombinant mouse IL-4. We found that the Rubisco-specific antibody titer in serum as well as in spleen cell culture supernatant was significantly enhanced in the IL-4 mouse. Most of the antibodies were of the IgG1 subclass. After hybridoma generation, Rubisco-specific antibodies were found in more than 95% of the wells tested compared to about 12% of the control mouse.