The First Human Epitope Map of the Alphaviral E1 and E2 Proteins Reveals a New E2 Epitope with Significant Virus Neutralizing Activity

Although the murine immune response to Venezuelan equine encephalitis virus (VEEV) is well-characterized, little is known about the human antibody response to VEEV. In this study we used phage display technology to isolate a panel of 11 VEEV-specfic Fabs from two human donors. Seven E2-specific and four E1-specific Fabs were identified and mapped to five E2 epitopes and three E1 epitopes. Two neutralizing Fabs were isolated, E2-specific F5 and E1-specific L1A7, although the neutralizing capacity of L1A7 was 300-fold lower than F5. F5 Fab was expressed as a complete IgG1 molecule, F5 native (n) IgG. Neutralization-escape VEEV variants for F5 nIgG were isolated and their structural genes were sequenced to determine the theoretical binding site of F5. Based on this sequence analysis as well as the ability of F5 to neutralize four neutralization-escape variants of anti-VEEV murine monoclonal antibodies (mapped to E2 amino acids 182–207), a unique neutralization domain on E2 was identified and mapped to E2 amino acids 115–119

Plasmid incompatibility: more compatible than previously thought?

It is generally accepted that plasmids containing the same origin of replication are incompatible. We have re-examined this concept in terms of the plasmid copy number, by introducing plasmids containing the same origin of replication and different antibiotic resistance genes into bacteria. By selecting for resistance to only one antibiotic, we were able to examine the persistence of plasmids carrying resistances to other antibiotics. We find that plasmids are not rapidly lost, but are able to persist in bacteria for multiple overnight growth cycles, with some dependence upon the nature of the antibiotic selected for. By carrying out the experiments with different origins of replication, we have been able to show that higher copy number leads to longer persistence, but even with low copy plasmids, persistence occurs to a significant degree. This observation holds significance for the field of protein engineering, as the presence of two or more plasmids within bacteria weakens, and confuses, the connection between screened phenotype and genotype, with the potential to wrongly assign specific phenotypes to incorrect genotypes

Solid wastes generation in the leather industry and its utilization for cleaner environment-A review

541-548Leather industry, one of the polluting industries because of generation of huge amount of liquid and solid wastes, also emits obnoxious smell because of degradation of proteinous material of skin and generation of gases such as NH3, H2S and CO2. Solid wastes are raw trimmings, fleshings, chrome shavings, buffing dusts and keratin wastes. Accumulation of these wastes lead to sludge problem and choking of treatment pipes and finally results in reduction in efficiency of treatment plant. Treatment of solid wastes also is not cost effective, posing economic burden to the tanners. Leather industry in the developing countries is facing lot of solid wastes problem and many tanneries closed for not meeting bio-chemical oxygen (BOD) demand and total dissolved solids (TDS) norms. The objective of this paper is to review the kinds of solid wastes generated in leather industry and the useful technologies developed to overcome the solid wastes problem

Development of an actinium-225 radioimmunoconjugate for targeted alpha therapy against SARS-CoV-2

Targeted alpha therapy offers unique opportunities for the treatment of tumours and infections. Here, we report the development of a new radioimmunoconjugate construct that targets SARS-CoV-2 infected cells, which act as viral reservoirs and promote virus replication and infection spread. The chosen antibody selectively binds to the ACE2-receptor binding domain of the spike protein, and prevents the protein binding to the receptor. Furthermore, the antibody has been radiolabelled with 225Ac, and the therapeutic performance of the resulting radioimmunoconjugate has been demonstrated in vitro against cells mimicking SARS-CoV-2 infection

Using T7 phage display to select GFP-based binders

Filamentous phage do not display cytoplasmic proteins very effectively. As T7 is a cytoplasmic phage, released by cell lysis, it has been prospected as being more efficient for the display of such proteins. Here we investigate this proposition, using a family of GFP-based cytoplasmic proteins that are poorly expressed by traditional phage display. Using two single-molecule detection techniques, fluorescence correlation spectroscopy and anti-bunching, we show that the number of displayed fluorescent proteins ranges from one to three. The GFP derivatives displayed on T7 contain binding loops able to recognize specific targets. By mixing these in a large background of non-binders, these derivatives were used to optimize selection conditions. Using the optimal selection conditions determined in these experiments, we then demonstrated the selection of specific binders from a library of GFP clones containing heavy chain CDR3 antibody binding loops derived from normal donors inserted at a single site. The selected GFP-based binders were successfully used to detect binding without the use of secondary reagents in flow cytometry, fluorescence-linked immunosorbant assays and immunoblotting. These results demonstrate that specific GFP-based affinity reagents, selected from T7-based libraries, can be used in applications in which only the intrinsic fluorescence is used for detection

Antibodies in proteomics I: generating antibodies

The explosion in genome sequencing, and in subsequent DNA array experiments, has provided extensive information on gene sequence, organization and expression. This has resulted in a desire to perform similarly broad experiments on all the proteins encoded by a genome. Panels of specific antibodies, or other binding ligands, will be essential tools in this endeavour. Because traditional immunization will be unlikely to generate antibodies in sufficient quantity, and of the required quality and reproducibility, in vitro selection methods will probably be used. This review--the first of two--examines the strategies available for in vitro antibody selection. The second review discusses the adaptation of these methods to high throughput and the uses to which antibodies, once derived, can be put

Antibodies in proteomics I: generating antibodies

11nonenoneBRADBURY A.; VELAPPAN N.; VERZILLO V.; OVECKA M.; OVECKA M.; CHASTEEN L.; SBLATTERO D.; MARZARI R.; LOU J.; SIEGEL R.; PAVLIK P.Bradbury, A.; Velappan, N.; Verzillo, V.; Ovecka, M.; Ovecka, M.; Chasteen, L.; Sblattero, Daniele; Marzari, Roberto; Lou, J.; Siegel, R.; Pavlik, P