Improvement of Fab expression by screening combinatorial synonymous signal sequence libraries

Background Antibody fragments can be expressed in Escherichia coli, where they are commonly directed to the periplasm via Sec pathway to enable disulphide bridge formations and correct folding. In order to transport antibody fragments to the periplasmic space via Sec pathway, they are equipped with N-terminal signal sequence. Periplasmic expression has many benefits but it's also subjected to many hurdles like inefficient translocation across the inner membrane and insufficient capacity of the translocation system. One solution to overcome these hurdles is a modulation of codon usage of signal sequence which has proved to be an efficient way of tuning the translocation process. Modulation of codon usage of signal sequences has been successfully employed also in improving the expression levels of antibody fragments, but unfortunately the effect of codon usage on the expression has not been thoroughly analyzed. Results In the present study we established three synonymous PelB signal sequence libraries by modulating codon usage of light chain and heavy chain PelB signal sequences of a Fab fragment. Each region (n-region, hydrophobic region and c-region) of the PelB signal sequence in the both chains of the Fab fragment in a bicistronic expression vector was mutated separately. We then screened for clones with improved expression profile. The best source for improved clones was the n-region library but in general, improved clones were obtained from all of the three libraries. After screening, we analyzed the effects of codon usage and mRNA secondary structures of chosen clones on the expression levels of the Fab fragment. When it comes to codon usage based factors, it was discovered that especially codon usage of fifth leucine position of the light chain PelB affects the expression levels of Fab fragment. In addition, we observed that mRNA secondary structures in the translation initiation regions of the light and heavy chain have an effect on expression levels as well. Conclusions In conclusion, the established synonymous signal sequence libraries are good sources for discovering Fab fragments with improved expression profile and obtaining new codon usage related information

Super-sensitive time-resolved fluoroimmunoassay for thyroid-stimulating hormone utilizing europium(III) nanoparticle labels achieved by protein corona stabilization, short binding time, and serum preprocessing.

Thyrotropin or thyroid-stimulating hormone (TSH) is used as a marker for thyroid function. More precise and more sensitive immunoassays are needed to facilitate continuous monitoring of thyroid dysfunctions and to assess the efficacy of the selected therapy and dosage of medication. Moreover, most thyroid diseases are autoimmune diseases making TSH assays very prone to immunoassay interferences due to autoantibodies in the sample matrix. We have developed a super-sensitive TSH immunoassay utilizing nanoparticle labels with a detection limit of 60 nU L(-1) in preprocessed serum samples by reducing nonspecific binding. The developed preprocessing step by affinity purification removed interfering compounds and improved the recovery of spiked TSH from serum. The sensitivity enhancement was achieved by stabilization of the protein corona of the nanoparticle bioconjugates and a spot-coated configuration of the active solid-phase that reduced sedimentation of the nanoparticle bioconjugates and their contact time with antibody-coated solid phase, thus making use of the higher association rate of specific binding due to high avidity nanoparticle bioconjugates. Graphical Abstract We were able to decrease the lowest limit of detection and increase sensitivity of TSH immunoassay using Eu(III)-nanoparticles. The improvement was achieved by decreasing binding time of nanoparticle bioconjugates by small capture area and fast circular rotation. Also, we applied a step to stabilize protein corona of the nanoparticles and a serum-preprocessing step with a structurally related antibody

Development of anti-immunocomplex specific antibodies and non-competitive time-resolved fluorescence immunoassay for the detection of estradiol

Detection of circulatory estradiol has widespread use in various clinical applications. Particularly, the use of estradiol-specific antibodies in immunoassays is routinely used, mainly due to the cost efficiency and simplicity of the sample handling process. However, the circulatory levels of estradiol can be extremely low in some conditions, and beyond the current detection limit of existing competitive immunoassays. We describe the generation of anti-immunocomplex specific antibodies derived from synthetic antibody repertoire and the development of high-performance non-competitive immunoassay for the detection of estradiol. Phage display selections were used to isolate new antibodies from synthetic antibody library with the use of existing estradiol specific Fab fragment. The found antibodies were consecutively used to set up a time-resolved fluorescence-based immunoassay (TRFIA), which can be used to detect estradiol with exceptional sensitivity and specificity. The limit of detection and EC50 were shown to be 3.0 pg mL−1 and 32.4 pg mL−1 respectively.</p

A Nanoparticle-Based Approach for the Detection of Extracellular Vesicles

The analysis of extracellular vesicles (EVs) typically requires tedious and time-consuming isolation process from bio-fluids. We developed a nanoparticle-based time resolved fluorescence immunoassay (NP-TRFIA) that uses biotinylated antibodies against the proteins of tetraspanin family and tumor-associated antigens for capturing EVs from urine samples and cell culture supernatants without the need for isolation. The captured-EVs were detected either with Eu3+-chelate or Eu3+-doped nanoparticle-based labels conjugated either to antibodies against the tetraspanins or lectins targeting the glycan moieties on EVs surface. The NP-TRFIA demonstrated specific capturing and detection of EVs by antibodies and lectins. Lectin-nanoparticle based assays showed 2–10 fold higher signal-to-background ratio compared with lectin-chelate assays. The nanoparticle assay concept allowed surface glycosylation profiling of the urine derived-EVs with lectins. It was also applied to establish an assay showing differential expression of tumor-associated proteins on more aggressive (higher ITGA3 on DU145- and PC3-EVs) compared to less aggressive (higher EpCAM on LNCaP-EVs) PCa- cell lines derived-EVs. This NP-TRFIA can be used as a simple tool for analysis and characterization of EVs in urine and cell culture supernatants. Such approach could be useful in identification of disease-specific markers on the surface of patient-derived urinary EVs

Archaic chaperone-usher pili self-secrete into superelastic zigzag springs

Adhesive pili assembled through the chaperone-usher pathway are hair-like appendages that mediate host tissue colonization and biofilm formation of Gram-negative bacteria(1-3). Archaic chaperone-usher pathway pili, the most diverse and widespread chaperone-usher pathway adhesins, are promising vaccine and drug targets owing to their prevalence in the most troublesome multidrug-resistant pathogens(1,4,5). However, their architecture and assembly-secretion process remain unknown. Here, we present the cryo-electron microscopy structure of the prototypical archaic Csu pilus that mediates biofilm formation of Acinetobacter baumannii-a notorious multidrug-resistant nosocomial pathogen. In contrast to the thick helical tubes of the classical type 1 and P pili, archaic pili assemble into an ultrathin zigzag architecture secured by an elegant clinch mechanism. The molecular clinch provides the pilus with high mechanical stability as well as superelasticity, a property observed for the first time, to our knowledge, in biomolecules, while enabling a more economical and faster pilus production. Furthermore, we demonstrate that clinch formation at the cell surface drives pilus secretion through the outer membrane. These findings suggest that clinch-formation inhibitors might represent a new strategy to fight multidrug-resistant bacterial infections

Effect of remdesivir post hospitalization for COVID-19 infection from the randomized SOLIDARITY Finland trial

We report the first long-term follow-up of a randomized trial (NCT04978259) addressing the effects of remdesivir on recovery (primary outcome) and other patient-important outcomes one year after hospitalization resulting from COVID-19. Of the 208 patients recruited from 11 Finnish hospitals, 198 survived, of whom 181 (92%) completed follow-up. At one year, self-reported recovery occurred in 85% in remdesivir and 86% in standard of care (SoC) (RR 0.94, 95% CI 0.47-1.90). We infer no convincing difference between remdesivir and SoC in quality of life or symptom outcomes (p > 0.05). Of the 21 potential long-COVID symptoms, patients reported moderate/major bother from fatigue (26%), joint pain (22%), and problems with memory (19%) and attention/concentration (18%). In conclusion, after a one-year follow-up of hospitalized patients, one in six reported they had not recovered well from COVID-19. Our results provide no convincing evidence of remdesivir benefit, but wide confidence intervals included possible benefit and harm.Peer reviewe