Development of a bioluminescent immunoassay based on Fc-specific conjugated antibody-nanoluciferase immunoreagents for determining aflatoxin B.

Aflatoxin B (AFB) is a potent carcinogen, and is among the most hazardous mycotoxins in agricultural products. Therefore, the development of sensitive and convenient detection methods for AFB is significant for food safety against mycotoxins. Herein, a bioluminescent enzyme immunoassay (BLEIA) was developed for ultrasensitive detection of AFB, based on the novel Fc-specific antibody-nanoluciferase (Ab-Nluc) conjugates which were fabricated using an IgG-binding protein-assisted photo-conjugation strategy.

Fabrication of cysteine-modified antibodies with Fc-specific conjugation for covalent and oriented immobilization of native antibodies.

Covalent and oriented immobilization of antibodies (Abs) can substantially improve the sensitivity and stability of solid-phase immunoassays. By modifying the natural Abs with functional groups that provide unique handles for further conjugation, Abs could be immobilized onto the solid matrices with uniform orientation. Herein, an effective approach for Fc-specific modification of Abs was developed for the oriented and covalent immobilization of Abs.

Development of site-specific antibody-conjugated immunoliposomes for sensitive detection of disease biomarkers.

Liposome-based immunoassay (LIA) is an attractive protocol for amplifying the detection signals because of the excellent ability of liposomes to encapsulate signal marker compounds. The antigen-binding activity of the conjugated antibodies on the liposomal surface is crucial for the specificity and sensitivity of LIA. We present here a general platform to ensure that antibodies can conjugate onto the surface of liposomes in a site-specific and oriented manner.

Directional immobilization of antibody onto magnetic nanoparticles by Fc-binding protein-assisted photo-conjugation for high sensitivity detection of antigen.

Immobilized antibodies with site-specific, oriented, and covalent pattern are of great significance to improve the sensitivity of solid-phase immunoassay. Here, we developed a novel antibody conjugation strategy that can immobilize antibodies in a directional and covalent manner. In this study, an IgG-Fc binding protein (Z domain) carrying a site-specific photo-crosslinker, p-benzoyl-L-phenylalanine, and a single C-terminal cysteine (Cys) handle was genetically engineered.

Fc-specific and covalent conjugation of a fluorescent protein to a native antibody through a photoconjugation strategy for fabrication of a novel photostable fluorescent antibody.

Fluorophore-antibody conjugates with high photobleaching resistance, high chemical stability, and Fc-specific attachment is a great advantage for immunofluorescence imaging. Here, an Fc-binding protein (Z-domain) carrying a photo-cross-linker (p-benzoylphenylalanine, Bpa) fused with enhanced green fluorescent protein (EGFP), namely photoactivatable Z-EGFP recombinant, was directly generated using the aminoacyl-tRNA synthetase/suppressor tRNA technique without any further modification. By employing the photoactivatable Z-EGFP, an optimal approach was successfully developed which enabled EGFP to site-selectively and covalently attach to native antibody (IgG) with approximately 90% conjugation efficiency.

Striatisporolide A, a butenolide metabolite from Athyrium multidentatum (Doll.) Ching, as a potential antibacterial agent.

The present study aimed to investigate the antibacterial activity of striatisporolide A (SA) against Escherichia coli (E. coli) and the underlying mechanism. Antibacterial activity was evaluated according to the inhibitory rate and zone of inhibition.

Site-specific, covalent immobilization of an engineered enterokinase onto magnetic nanoparticles through transglutaminase-catalyzed bioconjugation.

Enterokinase (EK) is one of the most popular enzymes for the in vitro cleavage of fusion proteins due to its high degree of specificity for the amino-acid sequence (Asp)-Lys. Enzyme reusability is desirable for reducing operating costs and facilitating the industrial application of EK. In this work, we report the controlled, site-specific and covalent cross-linking of an engineered EK on amine-modified magnetic nanoparticles (NH-MNPs) via microbial transglutaminase-catalyzed bioconjugation for the development of the oriented-immobilized enzyme, namely, EK@NH-MNP biocatalyst.

Fc-specific biotinylation of antibody using an engineered photoactivatable Z-Biotin and its biosensing application.

The development of a site-specific and covalent attachment methodology is crucial for antibody-biotin conjugates to preserve the antigen-binding ability of antibodies and yield homogeneous products. In this study, an engineered photoactivatable Z-domain variant [an UV-active amino acid benzoylphenylalanine (Bpa) was genetically incorporated into the Z-domain] carrying one biotin molecule (Z-Biotin) was prepared by employing aminoacyl-tRNA synthetase/suppressor tRNA and Avitag/BirA techniques. The site-specific and covalent attachment of IgG-biotin conjugates, viz.

Site-specific, covalent immobilization of BirA by microbial transglutaminase: A reusable biocatalyst for in vitro biotinylation.

A facile approach for the production of a reusable immobilized recombinant Escherichia coli biotin ligase (BirA) onto amine-modified magnetic microspheres (MMS) via covalent cross-linking catalyzed using microbial transglutaminase (MTG) was proposed in this study. The site-specifically immobilized BirA exhibited approximately 95% of enzymatic activity of the free BirA, and without a significant loss in intrinsic activity after 10 rounds of recycling (P > 0.05).

An efficient protocol to enhance the extracellular production of recombinant protein from Escherichia coli by the synergistic effects of sucrose, glycine, and Triton X-100.

Targeting recombinant proteins at highly extracellular production in the culture medium of Escherichia coli presents a significant advantage over cytoplasmic or periplasmic expression. In this work, a recombinant protein between ZZ protein and alkaline phosphatase (rZZ-AP) was constructed. Because rZZ-AP has the IgG-binding capacity and enzymatic activity, it can serve as an immunoreagent in immunoassays.

Formulation, optimization, and pharmacodynamic evaluation of chitosan/phospholipid/β-cyclodextrin microspheres.

Cholinergic neurotransmission loss is the main cause of cognitive impairment in patients with Alzheimer's disease. Phospholipids (PLs) play an essential role in memory and learning abilities. Moreover, PLs act as a source of choline in acetylcholine synthesis.

Site-specific covalent attachment of an engineered Z-domain onto a solid matrix: an efficient platform for 3D IgG immobilization.

Immobilized antibodies with oriented and homogeneous patterns are crucial to solid-phase molecular recognition assay. Antibody binding protein-based immobilization can effectively present the desired antibodies. However, steadily installing the stromatoid protein with site-specific attachment manner onto a matrix surface remains to be elucidated.

Development of an efficient signal amplification strategy for label-free enzyme immunoassay using two site-specific biotinylated recombinant proteins.

Constructing a recombinant protein between a reporter enzyme and a detector protein to produce a homogeneous immunological reagent is advantageous over random chemical conjugation. However, the approach hardly recombines multiple enzymes in a difunctional fusion protein, which results in insufficient amplification of the enzymatic signal, thereby limiting its application in further enhancement of analytical signal. In this study, two site-specific biotinylated recombinant proteins, namely, divalent biotinylated alkaline phosphatase (AP) and monovalent biotinylated ZZ domain, were produced by employing the Avitag-BirA system.

Well-oriented ZZ-PS-tag with high Fc-binding onto polystyrene surface for controlled immobilization of capture antibodies.

The site specificity and bioactivity retention of antibodies immobilized on a solid substrate are crucial requirements for solid phase immunoassays. A fusion protein between an immunoglobulin G (IgG)-binding protein (ZZ protein) and a polystyrene-binding peptide (PS-tag) was constructed, and then used to develop a simple method for the oriented immobilization of the ZZ protein onto a PS support by the specific attachment of the PS-tag onto a hydrophilic PS. The orientation of intact IgG was achieved via the interaction of the ZZ protein and the constant fragment (Fc), thereby displayed the Fab fragment for binding antigen.

Immobilization of unraveled immunoglobulin G using well-oriented ZZ-His protein on functionalized microtiter plate for sensitive immunoassay.

Highly efficient protein immobilization is extremely crucial for solid-phase immunoassays. We present a strategy for oriented immobilization of functionally intact immunoglobulin G (IgG) on a polystyrene microtiter plate via iminodiacetic acid (IDA)-Ni(2+) and ZZ-His protein interaction. We immobilized a ZZ-EAP (Escherichia coli alkaline phosphatase)-His fusion protein, which exhibits Fc binding, His tag, and intrinsic AP activities, and analyzed it against the interaction between rabbit IgG anti-horseradish peroxidase (anti-HRP) and its binding partner HRP to investigate the specificity and efficacy of this method.

Comparative characterization of recombinant ZZ protein-alkaline phosphatase and its application in enzyme immunoassays.

A functional fusion protein, which consists of an antibody and an enzyme that can be used in enzyme immunoassays, has been constructed. However, a quantitative comparison of the characteristics of fusion proteins and chemical conjugates of the parents, which are functionally produced in a uniform microbial system, has not been adequately achieved. In this study, a fusion protein between the ZZ protein and Escherichia coli alkaline phosphatase (AP) and the parental ZZ protein and AP for chemical conjugate was functionally produced in the same bacterial system.

Preparation of a bio-immunoreagent between ZZ affibody and enhanced green fluorescent protein for immunofluorescence applications.

In the present study, we constructed plasmid pUC-ZZ-EGFP to express Pro-ZZ-EGFP using ZZ peptide (a synthetic artificial IgG-Fc-fragment-binding protein derived from the B domain of staphylococcal protein A) and enhanced green fluorescent protein (EGFP). Without induction with isopropyl-β-D: -thiogalactopyranoside, the chimeric protein was effectively expressed in Escherichia coli HB101. Its affinity constant binding IgG was 2.

Effect of Glycine and Triton X-100 on secretion and expression of ZZ-EGFP fusion protein.

Two-factor and three-level fractional factorial design was employed for evaluation of the effect of Glycine and Triton X-100 on the secretion and expression of ZZ-EGFP fusion proteins. Varying contents of glycine (0%, 1%, 2%) and Triton X-100 (0%, 1%, 2%) were added into shaking flasks, respectively, and supplied with appropriate volume of ampicillin (total 9 combinations; group at concentration zero serving as control) to promote more ZZ-EGFP diffuse into liquid culture medium. Fluorescent intensity in the culture supernatant was detected.

Expression and secretion of recombinant ZZ-EGFP fusion protein by the methylotrophic yeast Pichia pastoris.

We constructed a fusion protein ZZ-EGFP by fusing the ZZ domains of staphylococcal protein A (SpA) and enhanced green fluorescent protein (EGFP). ZZ-EGFP was secreted in the yeast, Pichia pastoris, with a hexahistidine tag. Its expression level was determined by measuring the fluorescence of EGFP.

1,2-Bis(p-tolyl-sulfon-yl)hydrazine.

In the title compound, C(14)H(16)N(2)O(4)S(2), the dihedral angle between the aromatic ring planes is 76.8 (3)° and the S-N-N-S torsion angle is 122.5 (3)°.

[Study on correlation between relative density and kinematical viscosity of concentration for Yuxianling granules].

Objective: To study the relation between relative density and kinematical viscosity of the concentration for Yuxianling granules.

Method: The relative density and kinematical viscosity by alkali burette of the concentration were investigated. The connection between kinematical viscosity and spray drying, also and temperature of the concentration was respected.

[The extraction, purification and assaying of Gynostemma pentaphyllum polysaccharides].

By orthogonal design, and considering extracting efficiency and cost, optimizing the extract method of Gynostemma pentaphyllum polysaccharides. We purified the crude Gynostemma pentaphyllum polysaccharides initially, and assayed the polysaccharides content of Gynostemma pentaphyllum polysccharides. The content of Gynostemma pentaphyllum polysaccharides was sigificantly higher than the predecessor.