Preparation and identification of chicken egg yolk immunoglobulins against human enterovirus 71 for diagnosis of hand-foot-and-mouth disease.

Human enterovirus 71 (EV71) is one of the major pathogens that causes hand-foot-and mouth disease, and there is an urgent need for rapid diagnosis of EV71 virus infection for early antiviral treatment. The aim of this study was to prepare chicken egg yolk immunoglobulin Y (IgY) for the diagnosis of enterovirus type 71 infection. The antibodies were raised by intramuscular immunization of laying hens with inactivated human EV71 and isolated from the egg yolk by multiple steps of polyethylene glycol 6000 extraction. The average concentration of IgY antibody was 26.60 mg/mL during the whole immunization. After the first immunization, the IgY titer gradually increased, and reached the peak on the 55th days. Meanwhile, the use of western blotting test demonstrated that specific IgY binds specifically to capsid proteins VP2 and VP3 of EV71 virus. Furthermore, a facile one-step method based on turn-on fluorescence sensing was developed by using IgY antibodies, which can detect EV71 virus at low concentrations of 104 PFU/mL and was 94.44% coincidence with RT-PCR in 30 clinical samples. These findings indicate that EV71-IgY antibodies are an easily prepared and rich source of antibodies that offers a potential alternative strategy for routine screening of EV71 infection.

Correcting the Relative Bias of Light Obscuration and Flow Imaging Particle Counters.

PURPOSE: Industry and regulatory bodies desire more accurate methods for counting and characterizing particles. Measurements of proteinaceous-particle concentrations by light obscuration and flow imaging can differ by factors of ten or more. METHODS: We propose methods to correct the diameters reported by light obscuration and flow imaging instruments. For light obscuration, diameters were rescaled based on characterization of the refractive index of typical particles and a light scattering model for the extinction efficiency factor. The light obscuration models are applicable for either homogeneous materials (e.g., silicone oil) or for chemically homogeneous, but spatially non-uniform aggregates (e.g., protein aggregates). For flow imaging, the method relied on calibration of the instrument with silica beads suspended in water-glycerol mixtures. RESULTS: These methods were applied to a silicone-oil droplet suspension and four particle suspensions containing particles produced from heat stressed and agitated human serum albumin, agitated polyclonal immunoglobulin, and abraded ethylene tetrafluoroethylene polymer. All suspensions were measured by two flow imaging and one light obscuration apparatus. Prior to correction, results from the three instruments disagreed by a factor ranging from 3.1 to 48 in particle concentration over the size range from 2 to 20 µm. Bias corrections reduced the disagreement from an average factor of 14 down to an average factor of 1.5. CONCLUSIONS: The methods presented show promise in reducing the relative bias between light obscuration and flow imaging.

Structure and mechanism of cysteine peptidase gingipain K (Kgp), a major virulence factor of Porphyromonas gingivalis in periodontitis.

Cysteine peptidases are key proteolytic virulence factors of the periodontopathogen Porphyromonas gingivalis, which causes chronic periodontitis, the most prevalent dysbiosis-driven disease in humans. Two peptidases, gingipain K (Kgp) and R (RgpA and RgpB), which differ in their selectivity after lysines and arginines, respectively, collectively account for 85% of the extracellular proteolytic activity of P. gingivalis at the site of infection. Therefore, they are promising targets for the design of specific inhibitors. Although the structure of the catalytic domain of RgpB is known, little is known about Kgp, which shares only 27% sequence identity. We report the high resolution crystal structure of a competent fragment of Kgp encompassing the catalytic cysteine peptidase domain and a downstream immunoglobulin superfamily-like domain, which is required for folding and secretion of Kgp in vivo. The structure, which strikingly resembles a tooth, was serendipitously trapped with a fragment of a covalent inhibitor targeting the catalytic cysteine. This provided accurate insight into the active site and suggested that catalysis may require a catalytic triad, Cys(477)-His(444)-Asp(388), rather than the cysteine-histidine dyad normally found in cysteine peptidases. In addition, a 20-Å-long solvent-filled interior channel traverses the molecule and links the bottom of the specificity pocket with the molecular surface opposite the active site cleft. This channel, absent in RgpB, may enhance the plasticity of the enzyme, which would explain the much lower activity in vitro toward comparable specific synthetic substrates. Overall, the present results report the architecture and molecular determinants of the working mechanism of Kgp, including interaction with its substrates.

Addition of sodium malonate alters the morphology and increases the critical flux during tangential flow filtration of precipitated immunoglobulins.

Recent studies have demonstrated that one can control the packing density, and in turn the filterability, of protein precipitates by changing the pH and buffer composition of the precipitating solution to increase the structure/order within the precipitate. The objective of this study was to examine the effect of sodium malonate, which is known to enhance protein crystallizability, on the morphology of immunoglobulin precipitates formed using a combination of ZnCl2 and polyethylene glycol. The addition of sodium malonate significantly stabilized the precipitate particles as shown by an increase in melting temperature, as determined by differential scanning calorimetry, and an increase in the enthalpy of interaction, as determined by isothermal titration calorimetry. The sodium malonate also increased the selectivity of the precipitation, significantly reducing the coprecipitation of DNA from a clarified cell culture fluid. The resulting precipitate had a greater packing density and improved filterability, enabling continuous tangential flow filtration with minimal membrane fouling relative to precipitates formed under otherwise identical conditions but in the absence of sodium malonate. These results provide important insights into strategies for controlling precipitate morphology to enhance the performance of precipitation-filtration processes for the purification of therapeutic proteins.

[The development of polymer immunoglobulin preparations to identify different serovars legionella pneumophilia in reaction of slide-agglutination].

The article deals with the results of study targeted to develop polymer diagnostic preparation to identify epidemically significant serogroups Legionella pneumophilia. The preparation combines rate of record (1-5 min) of reaction of paragglutinining preparations with color visualization and demonstrative of reaction of volume agglomeration with polymer diagnosticums. The specially synthesized polymer microspheres were sensibilized with serums enriched with antibodies to lipopolysaccharide of corresponding serovar L. pneumophilia. The derived immunoglobulin diagnostic preparations detect agent of legionellesis in the reaction of slide-agglutination on glass during 1-5 min. The polymer diagnostic preparations provide positive reaction with culture of corresponding serovar and no reaction with other gomologic and geterologic agents of infectious diseases.

[Study on the stability of chicken egg yolk immunoglobulin (IgY) modified with mPEG].

The objective of the present paper was to study the effect of monomethoxypolyethlene glycol (mPEG) modification on the stability of chicken IgY and compare the stability of the modification products by Fourier transform infrared spectroscopy (FTIR), CD spectrooscopy and fluorescence spectroscopy. NHS-mPEG was used to modify IgY after mPEG was activated with N-hydroxysuccinimide (NHS). The optimal reaction condition for modification was 1:10 molar rate of IgY to mPEG at pH 7, reaction for 1 h, and the product was obtained with modification rate of 20.56% and activity reservation of 87. 62%. In addition, the thermal and pH stability of IgY and mPEG-IgY was compared by spectroscopic methods. The results showed that the alpha-helix, beta-sheet, beta-turn, and random content of IgY changed from 14.5%, 42.1%, 6.2% and 37.2% to 1.6%, 55.25%, 5.8% and 37.5%, while mPEG changed from 12.9%, 42.7%, 6.3% and 38. 1% to 3.1%, 50.5%, 7.2% and 39.2%, respectively, after incubating for 120 min at 70 degrees C. For the treatment with acid-base, similarly, the structure changes of mPEG-IgY were smaller than IgY. Thus, it is indicated that IgY modified by mPEG had greater stable properties.

A chromatographically purified human TGF-ß1 fraction from virally inactivated platelet lysates.

BACKGROUND AND OBJECTIVES: TGF-ß1 exerts important physiological functions in osteogenesis and chondrogenesis and may be of therapeutic interest. The aim of this work was to develop a scalable purification process of TGF-ß1 from virally inactivated human platelets. STUDY DESIGN AND METHODS: Apheresis platelet concentrates (N=12) were solvent/detergent (S/D) treated (1% TnBP/1% Triton X-45; 31°C) and the resulting platelet lysates were clarified by oil extraction and centrifugation, then chromatographed on an anion-exchange DEAE-Sepharose Fast-Flow column equilibrated in a PBS buffer, pH 7.5. The column was washed to eliminate unbound proteins and the S/D agents. Bound proteins were eluted using a 1 M NaCl-PBS buffer pH 7.5 (DEAE-eluate). The content in TGF-ß1, PDGF-AB, VEGF, IGF-1, EGF, and b-FGF was measured by ELISA. Proteins, lipids, and S/D agents were assessed. Protein profile was determined by SDS-PAGE under reduced or non-reduced conditions. RESULTS: Most proteins, including albumin and immunoglobulins G, A, and M did not bind to the DEAE column as evidenced also by SDS-PAGE. Essentially all PDGF, VEGF, and IGF were in the breakthrough. The DEAE-eluate contained close to 60% of the TGF-ß1 at a mean concentration of about 102 ng/ml, whereas EGF, b-FGF were at about 0.72 and 0.18 ng/ml, respectively. The content in TnBP and Triton X-45 was <2 ppm. CONCLUSION: A fraction enriched in TGF-ß1 can be prepared from virally inactivated human platelet lysates using an easily scale process. Its interest in regenerative medicine and cell therapy will be evaluated in further studies.

Positive response to surfactants on the interfacial behavior and aggregation stability of Fab fragments from yolk immunoglobulin.

The antigen binding fragment (Fab) is pepsin-digested product from egg yolk immunoglobulin (IgY), which shows lower immunogenicity and higher antibacterial activity. However, it limited the application of Fab due to the spontaneous adsorption and aggregation at the air-liquid interface. The present work is to investigate the effect of surfactants polysorbate 20 (PS20), poloxamer 188 (P188), and polyethylene glycol (PEG) on the aggregation stability of Fab of IgY. The results confirmed the positive role of surfactants in improving Fab stability. PS20 could effectively prevent the generation of Fab aggregates (DLS and light-obscuration analysis). It could also distinctly increase the internal hydrophobicity level, fortify the surface charge by altering the molecular conformational characteristics of Fab. The results of CLSM and surface tension demonstrated that P188 and PEG were co-adsorbed with Fab at the air-liquid interface and inhibited the formation of aggregation. PS20 competitively adsorbed in the gap between Fab molecules to inhibit the formation of aggregates. These findings would give an in-depth understanding of protein aggregation behavior influenced by surfactants and provide a theoretical basis for the development of functional food based on Fab active fragments.

Cell-Specific Delivery Using an Engineered Protein Nanocage.

Nanoparticle-based delivery systems have shown great promise for theranostics and bioimaging on the laboratory scale due to favorable pharmacokinetics and biodistribution. In this study, we examine the utility of a cage-forming variant of the protein lumazine synthase, which was previously designed and evolved to encapsulate biomacromolecular cargo. Linking antibody-binding domains to the exterior of the cage enabled binding of targeting immunoglobulins and cell-specific uptake of encapsulated cargo. Protein nanocages displaying antibody-binding domains appear to be less immunogenic than their unmodified counterparts, but they also recruit serum antibodies that can mask the efficacy of the targeting antibody. Our study highlights the strengths and limitations of a common targeting strategy for practical nanoparticle-based delivery applications.

Local administration and enhanced release of bone metabolic antibodies from hydroxyapatite/chondroitin sulfate nanocomposite microparticles using zinc cations.

As a local delivery carrier of bone metabolic proteins, we have previously reported hydroxyapatite/chondroitin sulfate composite microparticles (HAp/ChS) and their formulation method using zinc cations (Zn), and the in vitro release properties of proteins from the microparticles. Herein, we report the release properties of model antibodies such as immunoglobulin (IgG), human IgG (hIgG), and denosumab (Dmab) from HAp/ChS using this formulation method. Adding Zn in the formulation of IgG loaded with HAp/ChS microparticles enhanced the release of antibodies from HAp/ChS in phosphate buffer saline. In addition, the biological activity of Dmab released from HAp/ChS formulated with Zn was significantly higher than that without Zn. These results suggest a possible beneficial effect on the treatment for local bone diseases. The sclerostin monoclonal antibody (Sclmab) promotes fracture healing. We prepared HAp/ChS microparticles loaded with Sclmab and locally administered the microparticles into a drilled hole in the distal femoral bone of young rats. After three weeks, the area of the newly formed osteoid around the drilled hole where HAp/ChS loaded with Sclmab and Zn was locally administered was significantly higher than that observed in the control group (normal saline). Thus, HAp/ChS microparticles and the formulation method of monoclonal antibodies using Zn could be useful in the treatment of local bone diseases.

Immunoglobulins on the surface of differently charged polymer nanoparticles.

The overall success of nanocarriers in biomedical applications depends on their interaction with different proteins in blood. Immunoglobulins as a major protein class of the blood proteome may considerably influence the identity of the nanocarriers in blood. However, there is a lack of knowledge about the specific details of the interaction mechanism between different immunoglobulins and nanocarriers. Therefore, the authors have investigated the interaction of different immunoglobulin classes-namely, immunoglobulin G, A, and M-with different polystyrene model nanoparticles. The authors report that immunoglobulin interaction with nanoparticles strongly depends on the immunoglobulin class and surface charge of the nanoparticles. Furthermore, upon adsorption on the nanoparticles' surfaces, aggregation processes and denaturation of immunoglobulins were observed. This highlights the importance of nanocarriers' design in order to prevent unfavorable denaturation and adsorption processes of immunoglobulins on nanoparticle surfaces.

Integrated microfluidic immunoassay for the rapid determination of clenbuterol.

An integrated microfluidic immunoassay system was established for high throughput analysis of clenbuterol. This system consisted of an integrated microchip and a linear confocal laser induced fluorescence (LIF) scanner. The microchip was composed of three layers: a fluidic channel layer, a PDMS membrane layer and a pneumatic control layer. The multi-layer chip was integrated with 36 pneumatic micro-valves and multiple micro-pumps to realize the flexible reagent delivery, facilitating the automatic assays with less consumption of samples and reduced analysis time. The homemade LIF scanner was able to simultaneously detect multi-channels and provide the potential capability of high throughput assays. The performance of the system was demonstrated by the determination of clenbuterol, one of the most widely used beta-agonists. Under the optimal conditions, the linear range and the limit of detection of clenbuterol were 0 approximately 5.0 ng mL(-1) and 0.088 ng mL(-1), respectively. The recovery rates determined with pig urine samples of 1.0 ng mL(-1) and 2.0 ng mL(-1) were 98.74% and 102.51% (n = 3), respectively. The total detection time was less than 30 min. The system had the potential application for rapid detection of multiple beta-agonists in clinical, pharmaceutical and chemical analyses.

Photoactivation of alkyl C-H and silanization: a simple and general route to prepare high-density primary amines on inert polymer surfaces for protein immobilization.

Surface modification through implanting functional groups has been demonstrated to be extremely important to biomedical applications. The usage of organic polymer phase is often required to achieve satisfactory results. However, organic surfaces usually have poor chemical reactivity toward other reactants and target biomolecules because these surfaces usually only consist of simple alkyl (C-H) and/or alkyl ether (ROR') structures. For the first time, we here report the potential to perform silanization techniques on alkyl polymer surface, which provide a simple, fast, inexpensive, and general method to decorate versatile functional groups at the molecular level. As an example, high-density primary amines could be obtained on a model polymer, polypropylene substrate, through the reaction between amine-capped silane, 3-aminopropyltriethoxysilane (APTES) and hydroxylated polypropylene surface. A model protein, immunoglobulin (IgG), could be effectively immobilized on the surface after transforming amines to aldehydes by the aldehyde-amine condensation reaction between glutaraldehyde (GA) and amines. The routes we report here could directly make use of the benefits from well-developed silane chemistry, and hereby are capable of grafting any functionalities on inert alkyl surfaces via changing the terminal groups in silanes, which should instantly stimulate the development of many realms such as microarrays, immunoassays, biosensors, filtrations, and microseparation.

High-molecular-weight polymers from dietary fiber drive aggregation of particulates in the murine small intestine.

The lumen of the small intestine (SI) is filled with particulates: microbes, therapeutic particles, and food granules. The structure of this particulate suspension could impact uptake of drugs and nutrients and the function of microorganisms; however, little is understood about how this suspension is re-structured as it transits the gut. Here, we demonstrate that particles spontaneously aggregate in SI luminal fluid ex vivo. We find that mucins and immunoglobulins are not required for aggregation. Instead, aggregation can be controlled using polymers from dietary fiber in a manner that is qualitatively consistent with polymer-induced depletion interactions, which do not require specific chemical interactions. Furthermore, we find that aggregation is tunable; by feeding mice dietary fibers of different molecular weights, we can control aggregation in SI luminal fluid. This work suggests that the molecular weight and concentration of dietary polymers play an underappreciated role in shaping the physicochemical environment of the gut. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Rapid Size-Based Protein Discrimination inside Hybrid Isoporous Membranes.

Owing to their unique morphology, isoporous membranes derived from block copolymers (BCPs) have rapidly advanced the process of macromolecular separation. In such separations, fouling is the most daunting challenge, affecting both the permeability and selectivity of high-performance isoporous membranes. To overcome this, we increase the hydrophilicity of nanostructured BCP isoporous membranes by incorporating hydrophilic polymer-grafted graphene oxide nanosheets into them. Due to the synergy of these two highly functional components, the hybrid isoporous membranes show pH-responsive and alcohol-gating behaviors, along with improved bactericidal capabilities. Leveraging the high permeability and selectivity behavior of BCP isoporous membranes together with the antifouling capabilities imparted by the polymer-grafted graphene oxide nanosheets, we achieved the highest separation factor (33) ever obtained during the ultrafiltration of the common blood proteins bovine serum albumin and immunoglobulin. This was accompanied by a 60% enhanced flux compared to that of the pristine BCP membranes during this challenging size-based separation of a protein mixture. We surmise that such fouling-resistant hybrid isoporous membranes with rationally functionalized filler materials can be used to replace existing membranes for specific energy-efficient bioseparation applications with improved performance.

NHS-ester functionalized poly(PEGMA) brushes on silicon surface for covalent protein immobilization.

Poly(PEGMA) homopolymer brushes were developed by atom transfer radical polymerization (ATRP) on the initiator-modified silicon surface (Si-initiator). Through covalent binding, protein immobilization on the poly(PEGMA) films was enabled by further NHS-ester functionalization of the poly(PEGMA) chain ends. The formation of polymer brushes was confirmed by assessing the surface composition (XPS) and morphology (atomic force microscopy (AFM), scanning electronic microscopy (SEM)) of the modified silicon wafer. The binding performance of the NHS-ester functionalized surfaces with two proteins horseradish peroxidase (HRP) and chicken immunoglobulin (IgG) was monitored by direct observation. These results suggest that this method which incorporates the properties of polymer brush onto the binding surfaces may be a good strategy suitable for covalent protein immobilization.

Serum immunoglobulin fused interferon-alpha inhibited tumor growth in athymic mice bearing colon 26 adenocarcinoma cells.

Interferon (IFN) has therapeutic potential for a wide range of infectious and proliferative disorders. However, the half-life of IFN is too short to have a stable therapeutic effect. To overcome this problem, serum immunoglobulin has been fused to IFN. In this study, the efficacy of serum immunoglobulin fused INFs (si-IFN1 and si-IFN2) was evaluated on athymic mice bearing colon 26 adenocarcinoma cells. Seven days after the implantation of tumor cells, each group of mice was injected once a week with si-IFN1 and si-IFN2 at two different concentrations (10 x : 30 microg/kg and 50 x : 150 microg/kg). A slight anti-tumoral effect was observed in all 10 x groups compared to the control. In the 50 x groups, however, si-IFN1 and si-IFN2 showed significant anti- tumoral effects compared to the control. To gain more information on the mechanisms associated with the decrease of tumor size, a Western blot assay of apoptosis-related molecules was performed. The protein expression of cytochrome c, caspase 9, 6, and 3 were increased by si-IFN1 and si-IFN2. These 2 IFNs also increased the expressions of p53, p21, Bax and Bad. Interestingly, si-IFN1 and si-IFN2 decreased the expression of VEGF-beta. Taken together, serum immunoglobulin fused IFNs increased therapeutic efficacy under current experimental condition.

Comparison of the effects of three kinds of IgYs, (normal, nanoliposomal and nanoparticle conjugated), which are produced against the small domains of DR5 protein on cancer cells.

Cancer treatment with several kinds of drugs, especially targets the apoptotic pathways nowadays. TNF-related apoptosis-inducing ligand (TRAIL) as one of the important members of death receptors, significantly trigger induction of apoptosis in cancer cells. Three conserved domains of Death receptor (DR5) protein extracellular domain, which are fortified cysteine, were chosen and chemically synthesised. Hens were immunised with nano-liposomal peptides, and as a result the purified Immunoglobulin (IgYs) remarkably killed the cancerous MCF7 cells. The flow cytometric assay, confirmed the apoptotic death. Among several kinds of carriers that were used in this research, the nano-liposomal and nanoparticle conjugated, both were acceptable choices for drug delivery. Furthermore, the IgY against DR5's small peptides with such carriers successfully reached the target and significantly killed the cancer cells via apoptosis.

Membrane adsorbers as purification tools for monoclonal antibody purification.

Downstream purification processes for monoclonal antibody production typically involve multiple steps; some of them are conventionally performed by bead-based column chromatography. Affinity chromatography with Protein A is the most selective method for protein purification and is conventionally used for the initial capturing step to facilitate rapid volume reduction as well as separation of the antibody. However, conventional affinity chromatography has some limitations that are inherent with the method, it exhibits slow intraparticle diffusion and high pressure drop within the column. Membrane-based separation processes can be used in order to overcome these mass transfer limitations. The ligand is immobilized in the membrane pores and the convective flow brings the solute molecules very close to the ligand and hence minimizes the diffusional limitations associated with the beads. Nonetheless, the adoption of this technology has been slow because membrane chromatography has been limited by a lower binding capacity than that of conventional columns, even though the high flux advantages provided by membrane adsorbers would lead to higher productivity. This review considers the use of membrane adsorbers as an alternative technology for capture and polishing steps for the purification of monoclonal antibodies. Promising industrial applications as well as new trends in research will be addressed.

Surface modification of plastic, glass and titanium by photoimmobilization of polyethylene glycol for antibiofouling.

Photoreactive poly(ethylene glycol) (PEG) was prepared and the polymer was photoimmobilized on organic, inorganic and metal surfaces to reduce their interaction with proteins and cells. The photoreactive PEG was synthesized by co-polymerization of methacrylate-PEG and acryloyl 4-azidobenzene. Surface modification was carried in the presence and the absence of a micropatterned photomask. It was then straightforward to confirm the immobilization using the micropatterning. Using the micropatterning method, immobilization of the photoreactive PEG on plastic (Thermanox), glass and titanium was confirmed by time-of-flight secondary ion mass spectroscopy and atomic force microscopy observations. The contact angle on an unpatterned surface was measured. Although the original surfaces have different contact angles, the contact angle on PEG-immobilized surfaces was the same on all surfaces. This result demonstrated that the surface was completely covered with PEG by the photoimmobilization. To assess non-specific protein adsorption on the micropatterned surface, horseradish peroxidase (HRP)-conjugated proteins were adsorbed. Reduced protein adsorption was confirmed by vanishingly small staining of HRP substrates on the immobilized regions. COS-7 cells were cultured on the micropatterned surface. The cells did not adhere to the PEG-coated regions. In conclusion, photoreactive PEG was immobilized on various surfaces and tended to reduce interactions with proteins and cells.