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Toxicogenomic and Toxicoproteomic Approaches for Biomarkers
Published in Anthony P. DeCaprio, Toxicologic Biomarkers, 2006
As with subtractive libraries, this approach also relies on subsequent subcloning and sequencing of individual bands to identify the specific mRNAs. It also requires amplification and comparison of treated versus control using 24 to 48 different combinations of specific primers to theoretically cover all possible mRNAs. And it has an additional drawback that, because short products are produced by the random primer PCR step—and in fact the sequencing gel is optimal for visualizing bands in the 300 to 1000 base pair range—one often finds that the same mRNA is represented in multiple bands on the gels. As a result, it is possible to pick and sequence several differentially expressed bands and find that they are different PCR products of the same gene. Thus, this approach has now largely been replaced by DNA microarrays for studies in humans and several other major model organisms. However, it is still a potentially valuable approach in species for which there is little or no genomic information, to be used in lieu of or in addition to sequencing of expression libraries.
Functional Characterization Of Hybrid Ia antigens
Published in Soldano Ferrone, Chella S. David, Ia Antigens, 2019
Masao Kimoto, B. Beck, M. Shigeta, C. Garrison Fathman
The data presented above not only supported the existence of hybrid MLR stimulating determinants but additionally supported the existence of clones of responder cells present in strain A lymph node cells which could recognize such determinants on (B6A)F1 cells. In order to prove their existence, clones of alloreactive T cells were obtained by soft agar cloning techniques.14–16 Long-term cultures of alloreactive T cells were restimulated 24 to 36 hr prior to plating them in a 0.3% agar suspension which was poured over a base layer of 0.5% agar. Colonies were identified and picked from the soft agar utilizing drawn-out Pasteur pipettes 5 to 7 days later. Such colonies were replated in individual microtiter wells containing fresh irradiated stimulator cells and expanded by serial restimulation. Detailed methodology of such cloning and subcloning has been described.16 After suitable expansion to allow sufficient numbers of responder cells to be obtained, such soft agar colonies were assayed for their reactivity against stimulator cells from various strains. There were several types of reactivity patterns seen when these clones were assayed against a small panel of stimulator cells. For instance, clones derived from A(B6A)PRC or A(B6)PRC could be classified into several categories (Table 3). Clones originally labeled “Type 1” can be restimulated by the original stimulator cell [i.e., A(B6A)PRC clones are restimulated with (B6A)F1 stimulator cells; A(B6)PRC clones are stimulated with B6 stimulator cells]. Type 2 clones exhibited equal reactivities against both B6 and (B6A)F1 stimulator cells. Type 3 clones could be restimulated by several different stimulator cells, including third party stimulators against which they have not been primed. The existence of Type 1 clones derived from A(B6A)PRC seemed to confirm our hypothesis that there existed unique MLR stimulating determinants expressed on (B6A)F1 cells which were not expressed on cells of allogeneiic B6 stimulator cells or equal mixtures of A and B6 cells. One of the real anomalies observed in these experiments was the existence of Type 1 clones obtained from A(B6) bulk cultures. That is, there seem to exist unique homozygous MLR stimulating determinants expressed on B6 cells which are not expressed on heterozygous cells having B6 as one haplotype.16 Additionally, as shown by Type 4 clones, there seem to exist crossreactive MLR stimulating determinants shared between (B6A)F1 and third party stimulator cells not present on B6 cells.15 Current studies are in progress to try to identify the determinants that are shared between such crossreactive stimulator cells.
A Mycobacterial 65 kD Heat-Shock Protein and T Cells in Experimental Arthritis
Published in Thomas F. Kresina, Monoclonal Antibodies, Cytokines, and Arthritis, 2020
Willem van Eden, Claire J. P. Boog, Els J. M. Hogervorst, Marca H. M. Wauben, Ruurd van der Zee, Jan D. A. van Embden
Since mycobacterial species are notoriously difficult to grow in vitro, molecular cloning of their antigens seemed the solution for the development of diagnostics and vaccines. Thole et al. have been involved in cloning antigens of Mycobacterium bovis BCG, the strain used for tuberculosis vaccines, in Escherichia coli K12. A total of six different antigens were expressed as 30, 65, 70, 90, 95, and more than 100 kD molecular weights (11). From serology in mycobacterial diseases one antigen, the 65 kD protein, was already identified as a potent immunogen. By further subcloning of the latter coding gene an overproducing strain was obtained. From this strain the 65 kD could easily be purified and subsequently further characterized (12). By testing this particular protein in proliferation assays, the protein was found to stimulate A2, A2b, and A2c to an extent that exceeded even the stimulation obtained with the original selecting antigen, whole M. tuberculosis (13). All other mycobacterial recombinant proteins available to us at that time were found negative. Sequencing of the M. bovis BCG 65 kD protein coding gene revealed that it was composed of 540 amino acids and that it was identical with the M. tuberculosis 65 kD protein. Furthermore, the protein turned out to have a homology of more than 95% with its Mycobacterium leprae-derived counterpart. Thus, the antigen found responsible for triggering our arthritis T cells turned out to be a protein molecule conserved between various mycobacterial species. Furthermore, a panel of both polyclonal and monoclonal antibodies with specificity for this 65 kD mycobacterial protein was found to recognize, in Western blots, antigens of similar molecular weights in many other bacterial species. These species included arthritis-associated species, such as streptococci, Klebsiella, Shigella, Yersinia, gonococci, and Campylobacter. From these observations it was concluded that the 65 kD protein was a member of a family of proteins called “common antigen” (14). Further analysis revealed extensive sequence homology with the GroEL protein of E. coli, which is a so-called heat-shock or stress protein (15,16). The latter GroEL protein is a major essential protein present in virtually all bacterial cells. The expression of the GroEL gene(s) is upregulated under conditions that are stressful for the organism. The GroEL was also found to be a major protein present in mitochondria of eukaryotic cells, and also in plant chloroplasts. The reason for the evolutionary conservation of these molecules is probably related to their function as so-called molecular chaperones, which are essential to cell integrity, being involved in the assembly and possibly intracellular translocation of other multisubunit proteins.
Efficient production of bispecific antibody by FAST-IgTM and its application to NXT007 for the treatment of hemophilia A
Published in mAbs, 2023
Hikaru Koga, Takashi Yamano, Juan Betancur, Satoko Nagatomo, Yousuke Ikeda, Kazuki Yamaguchi, Yoshiaki Nabuchi, Kazuki Sato, Yuri Teranishi-Ikawa, Motohiko Sato, Hiroyuki Hirayama, Akira Hayasaka, Takuya Torizawa, Kenta Haraya, Zenjiro Sampei, Hirotake Shiraiwa, Takehisa Kitazawa, Tomoyuki Igawa, Taichi Kuramochi
Antibodies for FAST-Ig mutation screening were expressed transiently in Expi293F (Thermo Fisher Scientific) cells transfected with plasmids encoding immunoglobulin heavy chains and light chains, according to the manufacturer’s instructions (Thermo Fisher Scientific). Site-directed mutagenesis and sub-cloning into mammalian expression vectors were performed using an In-Fusion HD Cloning Kit (Clontech) or NEBuilder HiFi DNA Assembly Master Mix (New England BioLabs). Antibodies were purified by protein A affinity chromatography using a MabSelect PrismA (Cytiva). To assess the risk of mispaired species (Fig. S7D), plasmids encoding antibodies without FAST-Ig mutations were used for expression. Each HC contained ART-S3 mutations for enforcing the heterodimerization of HCs. Homodimeric antibodies (H1L1H1L1, H1L2H1L2, H2L1H2L1, H2L2H2L2) were prepared by co-transfecting the plasmids of each HC and LC of antibody, followed by protein A purification. To prepare the other six antibodies, pairs of parental homodimeric antibodies were separately prepared. These were mixed in 1:1 molar ratio and reconstituted in mildly reduced conditions (25 mmol/L 2-MEA), followed by dialysis to remove 2-MEA. Their BsAb yields were confirmed as >95% by CIEX analysis.
Characterization and initial demonstration of in vivo efficacy of a novel heat-activated metalloenediyne anti-cancer agent
Published in International Journal of Hyperthermia, 2022
Joy Garrett, Erin Metzger, Mark W. Dewhirst, Karen E. Pollok, John J. Turchi, Isabelle C. Le Poole, Kira Couch, Logan Lew, Anthony Sinn, Jeffrey M. Zaleski, Joseph R. Dynlacht
U-1 melanoma cells were cultured in McCoy’s 5 A Medium (Mediatech Inc./Corning Inc., Manassas, VA) with 10% iron-supplemented calf serum (ISCS)(Hyclone Laboratories, Logan, UT). PIG1 human melanocytes were cultured in M254 medium supplemented with Human Melanocyte Growth Supplement-2, L-glutamine (200 mM), and Antibiotic-Antimycotic (Life Technologies, Grand Island, NY). Authentication of U-1 cells was performed at IDEXX Biolytics (Columbia, MO). Isolation, cultivation and characterization of PIG1 cells were described previously by Le Poole et al. [34]. Briefly, neonatal human foreskin melanocytes isolated from skin samples were exposed to the LXSN16E5E7 retroviral vector, containing the E6 and E7 genes of human papilloma virus type 16 (HPV 16). Transfectants were selected based on geneticin resistance and genetic homogeneity ensured through subcloning. While PIG1 cells have growth potential at least twice that of primary normal human melanocytes, they retain melanocytic properties that are otherwise normal without an extensively altered phenotype. Importantly, they retain a 2n karyotype and will not form tumors in nude mice. Both cell lines were maintained in logarithmic growth in an incubator at 5% CO2 and 37 °C.
A novel multimeric IL15/IL15Rα-Fc complex to enhance cancer immunotherapy
Published in OncoImmunology, 2021
Hong Xu, Ilia N. Buhtoiarov, Hongfen Guo, Nai-Kong V. Cheung
Full-length ectodomain of IL15Rα (aa1-175) was fused to the CH2-CH3 of human IgG1 Fc region. Wild type (WT) and N72D mutated IL15 (MUT), in complex with either full length (FL) or Sushi domain (SU, aa1-66) of IL15Rα were made for comparison. These genes were synthesized by Genscript with appropriate flanking restriction enzyme sites, subcloned into a single two-segment mammalian expression vector, and used to transfect CHO-S cells (Invitrogen) for stable co-expression of protein complex and selection of high producers. 1 × 106 cells were transfected with 2.5 µg of linearized plasmid DNA by Nucleofection (Lonza) and then recovered in CD OptiCHO media supplemented with 8 mM L-glutamine (Invitrogen) for 2 days at 37°C in 6-well culture plate. Stable pools were selected first with 500 ug/ml hygromycin for approximately 2 weeks, single clones were then selected out with limited dilution. IL15/IL15Rα-Fc titer was determined by ELISA, where plates were coated with anti-human IL15 polyclonal goat IgG (R&D Systems) to capture the complex, and then detected with secondary goat anti-human IgG (Fc specific) (Southern Biotech). High expression clones with strong binding signals were selected for further subcloning. The complexes were purified using Protein A affinity chromatography.