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. A His-handle-modified antibody with Fc region-specific and covalent conjugation was first fabricated using a photoactivatable ZBpa-His tag that was engineered using the aminoacyl-tRNA synthetase/suppressor tRNA technique. Based on the high affinity between the His tag and divalent metal ions, the novel His-modified antibody was oriented onto the surface of nickel ion-modified liposomes encapsulating horseradish peroxidase. With the prostate-specific antigen as a model, the detection efficiency of the new immunoliposomes was evaluated by chemiluminescence immunoassay. The immunoliposomes exhibited a limit of detection of 0.2 pg mL-1, which was a six time improvement compared with that of the chemical-coupled antibody-liposome conjugates. Thus, the proposed immunoliposomes are expected to hold potential applications for the sensitive detection of various biomarkers in complicated serum samples.

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. Upon UV irradiation, the chimeric protein enables the Cys handle to couple with the native antibody in Fc-specific and covalent conjugation pattern, resulting in a novel thiolated antibody. Thus, an approach for the covalent, directional immobilization of antibodies to maleimide-modified magnetic nanoparticles (MNPs) was developed on the basis of the crosslinking between sulfhydryl and maleimide groups. The antibody-conjugated MNPs were applied in MNP-based enzyme-linked immunosorbent assay (ELISA) for the detection of carcinoembryonic antigen. The MNP-based ELISA presented a quantification linear range of 0.1-100 ng mL-1 and detection limit of 0.02 ng mL-1, which was approximately 100 times more sensitive than the traditional microplate ELISA (2.0 ng mL-1). Thus, the proposed antibody immobilization approach can be used in surface functionalization for the sensitive detection of various biomarkers.

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 ZBpa-EGFP recombinant, was directly generated using the aminoacyl-tRNA synthetase/suppressor tRNA technique without any further modification. By employing the photoactivatable ZBpa-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. After characterizing the Fc-specific and covalent manner of the EGFP-photoconjugated antibody, its excellent photobleaching resistance for immunofluorescence imaging was demonstrated in a model study by monitoring the toll-like receptor 4 (TLR4) expression in HepG2 cells. The proposed approach here for the preparation of a novel fluorescent antibody is available and reliable, which would play an important role in fluorescence immunoassay, and is expected to be extended to the generation of other biomolecule-photoconjugated antibodies, such as other fluorescent proteins for multiplex immunofluorescence imaging or reporter enzymes for highly sensitive enzyme immunoassays.Graphical abstract.

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. The antibacterial mechanism was investigated by analyzing alkaline phosphatase (AKP) activity and ATP leakage, protein expression, cell morphology and intracellular alterations in E. coli. The results demonstrated that SA exerted bacteriostatic effects on E. coli in vitro. AKP activity and ATP leakage analysis revealed that SA damaged the cell wall and cell membrane of E. coli. SDS­PAGE analysis indicated that SA notably altered the level of 10 and 35 kDa proteins. Scanning electron microscopy and transmission electron microscopy analyses revealed marked alterations in the morphology and ultrastructure of E. coli following treatment with SA. The mechanism underlying the antimicrobial effects of SA against E. coli may be attributed to its actions of disrupting the cell membrane and cell wall and regulation of protein level. The findings of the present study provide novel insight into the antimicrobial activity of SA as a potential natural antibacterial agent.

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)4-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 EKLC on amine-modified magnetic nanoparticles (NH2-MNPs) via microbial transglutaminase-catalyzed bioconjugation for the development of the oriented-immobilized enzyme, namely, EKLC@NH2-MNP biocatalyst. Upon the site-specific immobilization, approximately 90% EKLC enzymatic activity was retained, and the biocatalyst exhibited more than 85% of initial enzymatic activity regardless of storage or reusable stability over a month. The EKLC@NH2-MNP biocatalyst was further applied to remove the His tag-(Asp)4-Lys fusion partner from the His tag-(Asp)4-Lys-(GLP-1)3 substrate fusion protein, result suggested the EKLC@NH2-MNP possessed remarkable reusability, without a significant decrease of enzymatic activity over 10 cycles (P > 0.05). Supported by the unique properties of MNPs, the proposed EKLC@NH2-MNP biocatalyst is expected to promote the economical utilization of enterokinase in fusion protein cleavage.

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 (ZBpa-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. photo-biotinylated IgG, was successfully achieved after UV exposure by combining the inherent Fc-binding capability of the Z-domain with the formation of covalent bond by the photo-crosslinker. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay showed that more than 90% of IgGs conjugated with ZBpa-Biotin molecules suffered 3 h UV irradiation. Further pepsin digestion analysis confirmed that the ZBpa-Biotin was conjugated to the Fc fragment of IgG without interference. We took the tumor biomarker carcinoembryoic antigen (CEA) as model to evaluate the detection efficiency of the site-specific photo-biotinylated IgG in biosensing application using surface plasmon resonance (SPR) technology. The photo-biotinylated IgG coated surface gave a limit of detection (LOD) of 2 ng mL-1, is 5-fold lower than that of the randomly NHS-biotinylated IgG (10 ng mL-1). Given that the (strept)avidin-biotin complex is extensively used in immunoassays, the proposed method for biotinylated IgG provides a powerful approach to further expand related applications.

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). In addition, the immobilized BirA can be easily recovered from the solution via a simple magnetic separation. Thus, the immobilized BirA may be of general use for in vitro biotinylation in an efficient and economical manner.

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. However, only a very small portion of rZZ-AP is generally secreted into the aqueous medium under conventional cultivation procedure. Hence, we emphasized on the optimization of the culture procedures and attempted to dramatically enhance the yield of extracellular rZZ-AP from E. coli HB101 host cells by adding sucrose, glycine, and Triton X-100 in the culture medium. Results showed that the extracellular production of rZZ-AP in the culture medium containing 5% sucrose, 1% glycine, and 1% Triton X-100 was 18.6 mg/l, which was 18.6-fold higher than that without the three chemicals. And the ß-galactosidase activity test showed that the increased extracellular rZZ-AP was not due to cell lysis. Further analysis suggested a significant interaction effect among the three chemicals for the enhancement of extracellular production. Ultrastructural analysis indicated that the enhancement may be due to the influence of sucrose, glycine, and Triton X-100 on the periplasmic osmolality, permeability, or integrity of the cell wall, respectively. This proposed approach presents a simple strategy to enhance the extracellular secretion of recombinant proteins in the E. coli system at the process of cell cultivation.

Formulation, optimization, and pharmacodynamic evaluation of chitosan/phospholipid/ß-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. This study aimed to prepare and optimize the formulation of chitosan/phospholipid/ß-cyclodextrin (CTS/PL/ß-CD) microspheres that can improve cognitive impairment. The CTS/PL/ß-CD microspheres were prepared by spray drying, and optimized with an orthogonal design. These microspheres were also characterized in terms of morphology, structure, thermostability, drug loading, and encapsulation efficiency. The spatial learning and memory of rats were evaluated using the Morris water maze test, and the neuroprotective effects of the CTS/PL/ß-CD micro-spheres were investigated by immunohistochemistry. Scanning electron microscopic images showed that the CTS/PL/ß-CD microspheres were spherical with slightly wrinkled surfaces. Fourier transform infrared spectroscopy and differential scanning calorimetry proved that PLs formed hydrogen bonds with the amide group of CTS and the hydroxyl group of ß-CD. The learning and memory abilities of rats in the treated group significantly improved compared with those in the model group. Immunohistochemical analysis revealed that treatment with the CTS/PL/ß-CD microspheres attenuated the expression of protein kinase C-δ and inhibited the activation of microglias. These results suggest that the optimized microspheres have the potential to be used in the treatment of Alzheimer's disease.

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. In this study, we present an optimal protocol to tightly attach an immunoglobulin G (IgG)-binding protein (Z-domain) through covalent incorporation of Cys-tag and maleimide group onto polystyrene surface to guarantee site-specific, oriented, and irreversible attachment, resulting in a highly efficient platform for three-dimensional IgG immobilization. The actual IgG-binding characteristic of immobilized Z-Cys was investigated by employing affinity chromatography and size exclusion chromatography. And the efficacy and potential of this platform was demonstrated by applying it to the analysis of interaction between rabbit anti-HRP IgG and its binding partner HRP. The proposed approach may be an attractive strategy to construct high performance antibody arrays and biosensors given that the antibody is compatible with the Z-domain.

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. Through the high streptavidin (SA)-biotin interaction, the divalent biotinylated APs were clustered in the SA-biotin complex and then incorporated with the biotinylated ZZ. This incorporation results in the formation of a functional macromolecule that involves numerous APs, thereby enhancing the enzymatic signal, and in the production of several ZZ molecules for the interaction with immunoglobulin G (IgG) antibody. The advantage of this signal amplification strategy is demonstrated through ELISA, in which the analytical signal was substantially enhanced, with a 32-fold increase in the detection sensitivity compared with the ZZ-AP fusion protein approach. The proposed immunoassay without chemical modification can be an alternative strategy to enhance the analytical signals in various applications involving immunosensors and diagnostic chips, given that the label-free IgG antibody is suitable for the ZZ protein.

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. The interaction between rabbit IgG anti-horseradish peroxidase (anti-HRP) and its binding partner HRP was analyzed. Results showed that the oriented ZZ-PS-tag yielded an IgG-binding activity that is fivefold higher than that produced by the passive immobilization of the ZZ protein. The advantage of the proposed immunoassay strategy was demonstrated through an enzyme-linked immunosorbent assay, in which monoclonal mouse anti-goat IgG and HRP-conjugated rabbit F(ab')2 anti-goat IgG were used to detect goat IgG. The ZZ-PS-tag presented a tenfold higher sensitivity and a wider linear range than did the passively immobilized ZZ protein. The proposed approach may be an attractive strategy for a broad range of applications involving the oriented immobilization of intact IgGs onto PS supports, in which only one type of phi-PS (ZZ-PS-tag) surface is used.

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. A detailed examination of the ZZ-AP fusion protein and the effect of the ZZ-AP chemical conjugate on IgG affinity and enzymatic activity were performed. Compared with the parents, the equilibrium dissociation constant of ZZ-AP conjugate decreased by 32 % and catalytic activity decreased by 24 %, whereas the ZZ-AP fusion retained full parental activities and exhibited an approximately tenfold higher sensitivity than that of ZZ-AP conjugate in enzyme-linked immunosorbent assay. Thus, ZZ-AP fusion is a promising immunoreagent for IgG detection and a potential biolinker between antibodies and reporter enzymes (i.e., IgG-ZZ-AP fusion complex) in immunoassays.

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. We compared the IDA-Ni(2+)-(ZZ-His) method with ZZ-EAP random immobilization using sandwich enzyme-linked immunosorbent assay, and the results showed that the former method had an enhanced signal, 10-fold higher sensitivity, and a wider linear range. Thus, the proposed method allows a broad range of oriented immobilized functionally intact IgG antibodies on polystyrene plates using only one type of IDA-Ni(2+) chelate surface because the ZZ protein can bind to the Fc region of various IgGs.

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-ß-D: -thiogalactopyranoside, the chimeric protein was effectively expressed in Escherichia coli HB101. Its affinity constant binding IgG was 2.6 × 10(8) M(-1) obtained by competitive enzyme-linked immunosorbent assay, indicating that the ZZ peptide retains the native structure in Pro-ZZ-EGFP. The application of immunofluorescence assay for detecting the Mycoplasma pneumoniae IgG antibody, Pro-ZZ-EGFP, exhibited a good signal comparable in brightness and fluorescence pattern with the signal generated using the fluorescein isothiocyanate-labeled anti-human IgG. The result indicates that Pro-ZZ-EGFP possesses great potential for clinical immunofluorescence IgG test as an alternative versatile fluorescent antibody.

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. When the recombinant yeast cells in shake-flasks were induced with 0.5% methanol for 96 h, a maximum yield of 115 mg ZZ-EGFP/l was obtained. The resulting ZZ-EGFP fusion protein retained immunoglobulin G (IgG)-binding capacity and EGFP fluorescence. ZZ-EGFP was then used in immunofluorescence assays for detecting antinuclear antibodies (ANA); it produced a good signal that was comparable in its brightness and fluorescence pattern to that generated with fluorescein isothiocyanate (FITC)-labelled anti-human IgG. Thus, ZZ-EGFP showed great potential in immunological applications due to its ability to bind to various IgG from different animal sources.

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)°. In the crystal structure, mol-ecules form a chain structure by way of N-H⋯O hydrogen bonds.

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. A standard curve could be generated on the basis of fluorescent intensity and protein concentration. The expression level of ZZ-EGFP fusion proteins was estimated by checking the protein standard curve concentration fluorescene intensity. Results show that when the culture medium contains 2% Glycine and 1% Triton X-100, the expression level of ZZ-EGFP was able to be greatly increased. Further experiments revealed that absorbance value (A600) in the experiment group, whose culture medium contains 2% Glycine and 2% Triton X-100, is significantly lower than other groups in the present experiment. These results indicate that the culture medium containing appropriate quantity of Glycine and Triton X-100 is favourable to the secretion and expression level of ZZ-EGFP in gene-engineering bacteria Escherichia coli HB101.

[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. In the meantime, different mathematical models were simulated. RESULT: Kinematical viscosity is susceptible than relative density to the effect for spray drying, and the mathematical model is also set up accordingly. The result can offer the proper inlet temperature and kinematical viscosity for spray drying. CONCLUSIon The method in the experiment is simple, practical and manipulate easily. The study will provide the basis for extracted solution of compression and spray drying.

[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. It would provide conditions for the deep exploitation of Gynostemma pentaphyllum.