Biparatopic Nanobody-Based Immunosorbent for the Highly Selective Elimination of Tumor Necrosis Factor-α.

Removing the overexpressed TNF-α by hemoperfusion positively affects clinical treatments for diseases such as autoimmune disease and sepsis. However, clearance ratios of adsorbents targeting TNF-α were limited by the extremely low concentration of TNF-α (mostly <1000 ng/L in sepsis) and hydrophobic interactions. In this work, biparatopic nanobodies (NbC21) with a high affinity of 19.

SnoopLigase Enables Highly Efficient Generation of C-C-Linked Bispecific Nanobodies Targeting TNF-α and IL-17A.

Bispecific antibodies (bis-Nbs) have been extensively developed since the concept was devised over the decades. Taking advantage of the superior characteristics of nanobodies, bis-Nbs exhibit an emerging tendency to become the new generation of research and diagnostic tools. Traditional strategies to connect the homo- or heterogeneous monomers are commonly applied, but there are still technical issues to generate the bispecific molecules as efficiently as designed.

An engineered peptide tag-specific nanobody for immunoaffinity chromatography application enabling efficient product recovery at mild conditions.

Camelid-derived nanobody is emerging as a resourceful platform for developing immunoaffinity ligands for chromatography applications. Featured by high affinity and selectivity, BC2 nanobody (BC2-Nb), which can recognize a specific epitope tag (PDRKAAVSHWQQ, termed BC2T), is potential to be developed as a general tool for recombinant protein purification. However, excessively high affinity between binding partners makes the desorption of products less efficient and limits its application.

Nanobodies as solubilization chaperones for the expression and purification of inclusion-body prone proteins.

Here, we report a new protocol for enhancing the soluble expression of inclusion body (IB)-prone proteins in using nanobodies (Nbs) as a molecular-specific chaperone. The specific intracellular binding between the cognate-Nbs and the antigen is successfully achieved and enables the formation of a soluble Nb-antigen complex in . We further expand this method by adding an epitope tag (EPEA-tag) to the target proteins, and the anti-EPEA Nb was intended to act as the chaperone for binding with the EPEA tag.

Nanobody-loaded immunosorbent for highly-specific removal of interleukin-17A from blood.

Elimination of overproduced cytokines from blood can relieve immune system disorders caused by hypercytokinemia. Due to the central roles of interleukin-17A (IL-17A) plays in regulating the immunity and inflammatory responses in humans, here, a novel immunosorbent containing anti-IL-17A nanobodies (Nbs) was constructed for IL-17A removal from blood. The theoretical maximum adsorption capacity estimated from the Langmuir isotherm is up to 11.

Nanobody-Based high-performance immunosorbent for selective beta 2-microglobulin purification from blood.

Removing β2-microglobulin (β2M) from blood circulation is considered to be the most effective method to delay the occurrence of dialysis-related amyloidosis (DRA). The ideal extracorporeal β2M removal system should be cost-effective, highly specific and having a high capacity. However, the traditional technologies based on size exclusion do not have an adequate specificity, and alternative immunosorbents have limited applications due to low capacity and their high cost.

High Expression Achievement of Active and Robust Anti-β2 microglobulin Nanobodies via Hosts Selection.

Nanobodies (VHHs) overcome many of the drawbacks of conventional antibodies, and the related technologies represent state-of-the-art and advanced applications in scientific research, pharmaceuticals, and therapies. In terms of productivity and economic cost, the cytoplasmic expression of VHHs in () is a good process for their recombinant production. The cytoplasmic environment of the host is critical to the affinity and stability of the recombinant VHHs in soluble form, yet the effects have not been studied.

Generation and Application of Fluorescent Anti-Human β2-Microglobulin VHHs via Amino Modification.

The functionalization of VHHs enables their application in almost every aspect of biomedical inquiry. Amino modification remains a common strategy for protein functionalization, though is considered to be inferior to site-specific methods and cause protein property changes. In this paper, four anti-β2M VHHs were selected and modified on the amino group by NHS-Fluo.

One-step Preparation of a VHH-based Immunoadsorbent for the Extracorporeal Removal of β2-microglobulin.

Dialysis-related amyloidosis (DRA), which has been widely recognized to be associated with the accumulation of β2-microglobulin (β2-m) in blood, is one of the most common complications in patients receiving long-term dialysis treatment. The most significant side-effect of existing hemodialysis sorbents for the removal of β2-m from blood is the loss of vital proteins due to non-specific adsorptions. Although the traditional antibodies have the capability to specifically remove β2-m from blood, high cost limits their applications in clinics.

Oriented Immobilization and Quantitative Analysis Simultaneously Realized in Sandwich Immunoassay via His-Tagged Nanobody.

Despite the advantages of the nanobody, the unique structure limits its use in sandwich immunoassay. In this study, a facile protocol of sandwich immunoassay using the nanobody was established. In brief, β amyloid and SH2, an anti-β amyloid nanobody, were used as capture antibody and antigen, respectively.

A facile method to oriented immobilization of His-tagged BirA on Co-NTA agarose beads.

A facile and economical method was established for the oriented immobilization of biotin ligase (BirA) on Co-NTA sepharose through HO oxidation of Co and His-tag. His-tag of the BirA were designed at both N-terminal (His-BirA) and C-terminal (BirA-His), respectively. Immobilization of the His-BirA was performed, realized to 92.

Freezing-assisted synthesis of covalent C-C linked bivalent and bispecific nanobodies.

Bi-valent/specific antibodies are coming to the forefront of therapeutic and diagnostic applications for extending the functions of conventional antibodies. Nanobodies as building blocks, due to their small sizes, are prone to synthesizing these homo/hetero-dimers. However, the classical C-terminus to N-terminus (C-N) ligation manner for generating the dimer results in the inhibition of the antigen-binding capacity of the bivalent/specific antibodies.

Photoinitiated Polymerization-Induced Self-Assembly of Glycidyl Methacrylate for the Synthesis of Epoxy-Functionalized Block Copolymer Nano-Objects.

Herein, a novel photoinitiated polymerization-induced self-assembly formulation via photoinitiated reversible addition-fragmentation chain transfer dispersion polymerization of glycidyl methacrylate (PGMA) in ethanol-water at room temperature is reported. It is demonstrated that conducting polymerization-induced self-assembly (PISA) at low temperatures is crucial for obtaining colloidal stable PGMA-based diblock copolymer nano-objects. Good control is maintained during the photo-PISA process with a high rate of polymerization.

Polymerization-Induced Self-Assembly of Homopolymer and Diblock Copolymer: A Facile Approach for Preparing Polymer Nano-Objects with Higher-Order Morphologies.

Polymerization-induced self-assembly of homopolymer and diblock copolymer using a binary mixture of small chain transfer agent (CTA) and macromolecular chain transfer agent (macro-CTA) is reported. With this system, homopolymer and diblock copolymer were formed and chain extended at the same time to form polymer nano-objects. The molar ratio of homopolymer and diblock copolymer had a significant effect on the morphology of the polymer nano-objects.

Facile Preparation of CO -Responsive Polymer Nano-Objects via Aqueous Photoinitiated Polymerization-Induced Self-Assembly (Photo-PISA).

Carbon dioxide (CO )-responsive polymer nano-objects are prepared by photoinitiated reversible addition-fragmentation chain transfer dispersion polymerization of 2-hydroxypropyl methacrylate and 2-(dimethylamino)ethyl methacrylate (DMAEMA) in water at room temperature using a poly(poly(ethylene glycol) methyl ether methacrylate) macromolecular chain transfer agent. Kinetic studies confirm that full monomer conversions are achieved in all cases within 10 min of visible-light irradiation (405 nm, 0.5 mW cm ).

Alcoholic Photoinitiated Polymerization-Induced Self-Assembly (Photo-PISA): A Fast Route toward Poly(isobornyl acrylate)-Based Diblock Copolymer Nano-Objects.

We report a fast alcoholic photoinitiated polymerization-induced self-assembly (photo-PISA) formulation via photoinitiated RAFT dispersion polymerization of isobornyl acrylate (IBOA) in an ethanol/water mixture at 40 °C using a monomethoxy poly(ethylene glycol) (mPEG) based chain transfer agent. Polymerization proceeded rapidly via the exposure to visible light irradiation (405 nm, 0.5 mW/cm), and high monomer conversion (>95%) was achieved within 30 min.

Low-Temperature Synthesis of Thermoresponsive Diblock Copolymer Nano-Objects via Aqueous Photoinitiated Polymerization-Induced Self-Assembly (Photo-PISA) using Thermoresponsive Macro-RAFT Agents.

Photoinitiated reversible addition-fragmentation chain transfer (RAFT) dispersion polymerization of 2-hydroxypropyl methacrylate is conducted in water at low temperature using thermoresponsive copolymers of 2-(2-methoxyethoxy) ethyl methacrylate and oligo(ethylene glycol) methacrylate (Mn = 475 g mol(-1) ) as the macro-RAFT agent. Kinetic studies confirm that quantitative monomer conversion is achieved within 15 min of visible-light irradiation (405 nm, 0.5 mW cm(-2) ), and good control is maintained during the polymerization.