Competitive HRP-Linked Colorimetric Aptasensor for the Detection of Fumonisin B1 in Food based on Dual Biotin-Streptavidin Interaction

Fumonisin B1 (FB1) is the most prevalent and toxic form among fumonisin homologues which are produced by fusarium species and it contaminates various types of food products, posing serious health hazards for humans and animals. In this work, a colorimetric assay for the detection of FB1 has been developed based on competitive horseradish peroxidase (HRP)-linked aptamer and dual biotin-streptavidin interaction. In short, a biotinylated aptamer of FB1 was immobilized on the microplate by biotin-streptavidin binding; the complementary strand (csDNA) of the aptamer was ligated with HRP by biotin-streptavidin binding again to form a csDNA-HRP sensing probe, competing with FB1 to bind to the aptamer. The color change can be observed after the addition of chromogenic and stop solution, thereby realizing the visual detection of FB1. Under optimal conditions, good linearity was observed within the concentration range of 0.5 to 300 ng/mL, with a detection of limit of 0.3 ng/mL. This assay is further validated by spike recovery tests towards beer and corn samples, it provides a simple, sensitive and reliable method for the screening of FB1 in food samples and may be potentially used as an alternative to conventional assays.

Development of a nanobody tagged with streptavidin-binding peptide and its application in a Luminex fluoroimmunoassay for alpha fetal protein in serum

A nanobody/streptavidin-binding peptide fusion protein was developed and proved to be a very promising immunological diagnosis reagent for disease-related biomarkers.

A Generic Method for Fast and Sensitive Detection of Adeno-Associated Viruses Using Modified AAV Receptor Recombinant Proteins

Adeno-Associated Viruses (AAV) are widely used gene-therapy vectors for both clinical applications and laboratory investigations. The titering of different AAV preparations is important for quality control purposes, as well as in comparative studies. However, currently available methods are limited in their ability to detect various serotypes with sensitivity and convenience. Here, we took advantage of a newly discovered AAV receptor protein with high affinity to multiple AAV serotypes, and developed an ELISA-like method named “VIRELISA” (virus receptor-linked immunosorbent assay) by adopting fusion with a streptavidin-binding peptide (SBP). It was demonstrated that optimized VIRELISA assays exhibited satisfactory performance for the titering of AAV2. The linear range of AAV2 was 1 × 105 v.g. to 5 × 109 v.g., with an LOD (limit of detection) of 5 × 104 v.g. Testing of VIRELISA for the quantification of AAV1 was also successful. Our study indicated that a generic protocol for the quantification of different serotypes of AAVs was feasible, reliable and cost-efficient. The applications of VIRELISA will not only be of benefit to laboratory research due to its simplicity, but could also potentially be used for monitoring the circulation AAV loads both in clinical trials and in wild type infection of a given AAV serotype.

Artificial tethering of LC3 or p62 to organelles is not sufficient to trigger autophagy

The retention using selective hooks (RUSH) system allows to retain a target protein fused to green fluorescent protein (GFP) and a streptavidin-binding peptide (SBP) due to the interaction with a molar excess of streptavidin molecules (“hooks”) targeted to selected subcellular compartments. Supplementation of biotin competitively disrupts the interaction between the SBP moiety and streptavidin, liberating the chimeric target protein from its hooks, while addition of avidin causes the removal of biotin from the system and reestablishes the interaction. Based on this principle, we engineered two chimeric proteins involved in autophagy, namely microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B, best known as LC3) and sequestosome-1 (SQSTM1, best known as p62) to move them as SBP–GFP–LC3 and p62–SBP–GFP at will between the cytosol and two different organelles, the endoplasmic reticulum (ER) and the Golgi apparatus. Although both proteins were functional in thus far that SBP–GFP–LC3 and p62–SBP–GFP could recruit their endogenous binding partners, p62 and LC3, respectively, their enforced relocation to the ER or Golgi failed to induce organelle-specific autophagy. Hence, artificial tethering of LC3 or p62 to the surface of the ER and the Golgi is not sufficient to trigger autophagy.

A direct selection strategy for isolating aptamers with pH-sensitive binding activity

An aptamer reagent that can switch its binding affinity in a pH-responsive manner would be highly valuable for many biomedical applications including imaging and drug delivery. Unfortunately, the discovery of such aptamers is difficult and only a few have been reported to date. Here we report the first experimental strategy for generating pH-responsive aptamers through direct selection. As an exemplar, we report streptavidin-binding aptamers that retain nanomolar affinity at pH 7.4 but exhibit a ~100-fold decrease in affinity at pH 5.2. These aptamers were generated by incorporating a known streptavidin-binding DNA motif into an aptamer library and performing FACS-based screening at multiple pH conditions. Upon structural analysis, we found that one aptamer’s affinity-switching behavior is driven by a non-canonical G-A base-pair that controls its folding in a highly pH-dependent manner. We believe our strategy could be readily extended to other aptamer-target systems because it does not require a priori structural knowledge of the aptamer or the target.

BIOTINYLATED AND CHELATED POLY-L-LYSINE AS EFFECTOR FOR PRETARGETING IN CANCER THERAPY AND IMAGING

<p><strong>Objective: </strong>The aim of this study was to synthesise and evaluate polylysine-based effectors for pretargeted radioimmunotherapy and imaging. These molecules can readily be size-modified and charge-modified to decrease the renal uptake of radioactivity, which is often a major problem for small radiolabeled molecules. Several chelators and biotin molecules (for antibody-streptavidin-binding <em>in vivo</em>) are also easily incorporated into one structure because of the polylysine.</p><p><strong>Methods: </strong>The effectors were synthesised using poly-L-lysine, NHS-LC-biotin, CHX-A’’-DTPA or <em>p</em>-SCN-Bn-DOTA and succinic anhydride. They were characterised, labelled with ²¹³Bi for targeted α therapy, ⁶⁸Ga for PET and ¹¹¹In for SPECT, and evaluated <em>in vitro</em>. A kidney uptake study was performed as well with two different-sized ²¹³Bi-labeled effectors, to evaluate how the difference in size affects the renal filtration.</p><p><strong>Results: </strong>Radiochemical purities between 97.4±0.6 % and 99.6±0.1 % and decay-corrected yields of 80.2±2.4 % after purification were achieved with the radiolabeled molecules, as well as a specific activity of 7.6 × 10³GBq/µmol. The avidin binding capacity was 94.4±1.9%. The kidney uptake study demonstrated a reduction of renal absorbed dose by 80% when modifying the molecular size and charge.</p><p><strong>Conclusion: </strong>The synthesised polylysine-based effectors show potential for further <em>in vivo</em> evaluation in pretargeted radioimmunotherapy and imaging.</p>