Rapid production of a chimeric antibody-antigen fusion protein based on 2A-peptide cleavage and green fluorescent protein expression in CHO cells

To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.     

Ubiquitin-coated nanodiamonds bind to autophagy receptors for entry into the selective autophagy pathway

Selective macroautophagy/autophagy plays a pivotal role in the processing of foreign pathogens and cellular components to maintain homeostasis in human cells. To date, numerous studies have demonstrated the uptake of nanoparticles by cells, but their intracellular processing through selective autophagy remains unclear. Here we show that carbon-based nanodiamonds (NDs) coated with ubiquitin (Ub) bind to autophagy receptors (SQSTM1 [sequestosome 1], OPTN [optineurin], and CALCOCO2/NDP52 [calcium binding and coiled-coil domain 2]) and are then linked to MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) for entry into the selective autophagy pathway. NDs are ultimately delivered to lysosomes. Ectopically expressed SQSTM1-green fluorescence protein (GFP) could bind to the Ub-coated NDs. By contrast, the Ub-associated domain mutant of SQSTM1 (ΔUBA)-GFP did not bind to the Ub-coated NDs. Chloroquine, an autophagy inhibitor, prevented the ND-containing autophagosomes from fusing with lysosomes. Furthermore, autophagy receptors OPTN and CALCOCO2/NDP52, involved in the processing of bacteria, were found to be involved in the selective autophagy of NDs. However, ND particles located in the lysosomes of cells did not induce mitotic blockage, senescence, or cell death. Single ND clusters in the lysosomes of cells were observed in the xenografted human lung tumors of nude mice. This study demonstrated for the first time that Ub-coated nanoparticles bind to autophagy receptors for entry into the selective autophagy pathway, facilitating their delivery to lysosomes.     

Detection of Plasmodium falciparum DNA in a microtitration plate-based hybridization test

A rapid DNA-test, depending on the affinity based hybrid collection principle, was developed for the detection of Plasmodium falciparum DNA from clinical specimens. In this method, hybridization takes place in solution and the hybrids are collected onto a solid phase for measurement. Two probes are used, one labelled with an affinity tag (biotin) and the other with a detectable label (32P). In the present test a single oligonucleotide complementary to a 21-base pair sequence which is highly repeated in the parasite genome served both as capture and detector probe. The test is a 2-h hybridization performed in streptavidin coated microtitration plate wells, onto which the labelled hybrids simultaneously bind. The sensitivity of the assay with a crude erythrocyte lysate specimen was 1.6 x 10(9) repeat units corresponding to about 160 parasites in one microliter blood. The results allowed quantification of the repeat sequences and thus estimation of the degree of parasitemia in clinical specimens.     

Complementary Aluminum Nanopatch/Nanohole Arrays for Broad Palettes of Colors

Plasmonics - We investigate aluminum nanopatch/nanohole arrays surrounded by a dielectric material on plastic substrates for large area color printing. In this specific arrangement, metallic...     

Quality control in the determination of cortisol in plasma/serum by using, on every sample, two different three-step separation methods including ultrafiltration, restricted-access high-performance liquid chromatography and reversed-phase high-performance liquid chromatography, and contrasting results to immunoassays

Tests of HPLC columns with restricted access, polymer covered alumina, polymer, and different ODS phases showed that base-acid compatible ODS columns gave the best peak shapes of cortisol, internal standard, as well as of plasma/serum (P/S) matrix components. Further trials with cortisol in P/S showed that three separation steps were essential in order to obtain chromatographic data which were superior to immunoassay data. Also, sufficient confidence in results required determination of each sample with two newly developed separation methods: (a) pre-separation with a restricted access column, concentration of the desired cut with a 20 mm base-acid compatible ODS column, and analysis with a 250 mm column filled with the same ODS; (b) pre-separation with an ultrafilter followed by the last two steps in (a). For detection UV was preferred over fluorescence. This twin multistep chromatography showed that immunoassays were very treacherous in that they produced a spectrum of results ranging from good to untenable without any warning whatever about functionality. The measurement of official controls, with reference values derived via gas chromatography-isotope dilution mass spectrometry, also demonstrated the superiority of the double HPLC method.     

Membrane—vanadium interaction: A toxicokinetic evaluation

Vanadium is an important trace metal widely distributed in environment. Interaction of vanadate with skeletal muscle sarcolemma and basement membrane has been focussed. Scatchard analysis indicated the presence of more than one binding site for vanadate. Vanadate inhibits sarcolemmal and intestinal brush border membrane enzymes in a non-competitive manner. Membrane-vanadium interaction may lead to several structural and functional changes. The binding of vanadium to basement membrane may have some protective role.     

Isolation and characterization of a human hepatic epithelial-like cell line (AKN-1) from a normal liver

Summary The isolation and characterization of human liver cell lines are rather difficult due to limited material and poor growth in cell culture. In this report, we present the isolation, culture and characterization of a new epithelial-like liver cell line (AKN-1) with a heterogeneous cell population and many characteristics of the biliary epithelium. The AKN-1 cell line stained positively with antibodies to epithelial cytokeratin polypetides CK 8, 18, and 19. In addition, the cell line expressed the anti-human epithelial-related antigen (MOC-31), the human epithelial antigen (HEA), and the gamma-glutamyl transpeptidase, the hematopoietic growth factor, stem cell factor, and also its receptor, c-kit. The cell line failed to express albumin and factor 8 by immunohistochemistry. It did show, however, a twofold increase in 7-ethoxyresorufin-O-deethylase activity. Cytogenetic characterization revealed rare breakpoints in chromosome 2, which to our knowledge, have not yet been reported in liver cells.