[Screening and activity analysis of nanobodies against lymphocyte activation gene-3(LAG-3)].

Objective To generate the phage display nanobody library immunized by lymphocyte-activation gene 3 (LAG-3) and to validate the functional activity of obtained anti-LAG-3 nanobodies. Methods The peripheral blood cDNA library was isolated from the adult llama which was immunized by human LAG-3 protein. The nanobodies sequences were obtained by nested PCR and cloned into the phagemid vector pComb3XSS, then transformed into Escherichia coli XL1-Blue cells for library generation and quality analysis. Anti-LAG-3 specific nanobodies were screened by phage display and sequenced by next-generation sequencing. Nanobodies were cloned into pET-22b (+) vector and Escherichia coli BL21 (DE3) cells were used for protein expression. The proteins were purified by using the Prism A column, then HPLC-MS, ELISA, Western blot, and surface plasmon resonance technology (SPR) were performed to characterize the nanobodies. Results The library capacity of the nanobody phage immune library with great diversity was 7.20×108 CFU/mL. After four rounds of biopanning, three individual nanobodies with distinct amino acid sequences VHH-L1-3, VHH-L3-2 and VHH-L13-2 were picked. The purity of the purified nanobodies was more than 95%. All of these three nanobodies exhibited high binding affinities with recombinant human LAG-3 specifically, among which the KD value of VHH-L13-2 was 3.971×10-9 mol/L. VHH-L13-2 exhibited the inhibitory effects on the association of LAG-3 and its ligand FGL-1, and the half maximal inhibitory concentration (IC50) value was 15.58 nmol/L. Conclusion The anti-LAG-3 phage display nanobody library is generated successfully. The anti-LAG-3 nanobodies possess high specificity and binding affinity and exhibit the inhibitory effects on the association of LAG-3 and its ligand.

Research advances in treatment methods and drug development for rare diseases.

As the incidence of rare diseases increases each year, the total number of rare disease patients worldwide is nearly 400 million. Orphan medications are drugs used to treat rare diseases. Orphan drugs, however, are rare and patients often struggle to utilize them and expensive medications during treatment. Orphan drugs have been the focus of new drug research and development for both domestic and international pharmaceutical companies as a result of the substantial investment being made in the field of rare diseases. Clinical breakthroughs have been made in every field, from traditional antibodies and small molecule drugs to gene therapy, stem cell therapy and small nucleic acid drugs. We here review the therapeutic means of rare diseases and drug development of rare diseases to show the progress of treatment of rare diseases in order to provide a reference for clinical use and new drug development of rare diseases in China.

Silver Ions as Novel Coreaction Accelerator for Remarkably Enhanced Electrochemiluminescence in a PTCA-S2O82- System and Its Application in an Ultrasensitive Assay for Mercury Ions.

In this work, with the use of Ag(I) ion as robust coreaction accelerator for the enhancement of 3,4,9,10-perylenetetracarboxylic acid-peroxydisulfate (PTCA-S2O82-) system, a highly sensitive solid-state electrochemiluminescence (ECL)-biosensing platform was successfully designed for the detection of mercury ions (Hg2+). Specifically, a long guanine-rich (C-rich) double-stranded DNA (dsDNA) was generated by the target-Hg2+-controlled DNA machine that could amplify the ECL signal of the PTCA-S2O82- system by embedding the Ag(I) ion. Herein, the Ag(I) ion, as a coreaction accelerator, could first react with S2O82- to produce Ag(II) ion and a sulfate radical anion (SO4·-). Then, the accompanying Ag(II) ion could react with H2O to generate the reactive intermediate species (i.e., hydroxyl radical (OH·)), which could further accelerate the reduction of S2O82- to output more SO4·-. Moreover, the recycling of the Ag(I) ion and Ag(II) ion was easily achieved by the electrochemical reaction. Therefore, an avalanche-type reaction was triggered to generate massive amounts of SO4·-, which could react with the luminophore (PTCA) to achieve an extremely strong ECL signal. The ECL mechanism was investigated by ECL and cycle voltammetry (CV) and by the analysis of the fluorescence (FL), ECL, and electron-paramagnetic-resonance (EPR) spectra. As a result, the proposed solid-state ECL-biosensing platform for Hg2+ detection exhibited high sensitivity, with a linear range from 1 × 10-15 to 1 × 10-10 M and a detection limit of 3.3 × 10-16 M. Importantly, this work was the first to utilize a metal ion as a coreaction accelerator and provided a promising approach to improve the sensitivity of target analyses in ECL-biosensing fields.