Albumin Conjugate Research Articles

Recombinant Peptide Production Platform Coupled with Site-Specific Albumin Conjugation Enables a Convenient Production of Long-Acting Therapeutic Peptide.

The number of therapeutic peptides for human treatment is growing rapidly. However, their development faces two major issues: the poor yield of large peptides from conventional solid-phase synthesis, and the intrinsically short serum half-life of peptides. To address these issues, we investigated a platform for the production of a recombinant therapeutic peptide with an extended serum half-life involving the site-specific conjugation of human serum albumin (HSA). HSA has an exceptionally long serum half-life and can be used to extend the serum half-lives of therapeutic proteins and peptides. We used glucagon-like-peptide 1 (GLP-1) as a model peptide in the present study. A “clickable” non-natural amino acid—p-azido-l-phenylalanine (AzF)—was incorporated into three specific sites (V16, Y19, and F28) of a GLP-1 variant, followed by conjugation with HSA through strain-promoted azide–alkyne cycloaddition. All three HSA-conjugated GLP-1 variants (GLP1_16HSA, GLP1_19HSA, and GLP1_28HSA) exhibited comparable serum half-lives in vivo. However, the three GLP1_HSA variants had different in vitro biological activities and in vivo glucose-lowering effects, demonstrating the importance of site-specific HSA conjugation. The platform described herein could be used to develop other therapeutic peptides with extended serum half-lives.

Production and characterization of monoclonal antibodies against GII.6 norovirus virus-like particles

In this study we generated and characterized a series of monoclonal antibodies (mAbs) against GII.6 norovirus (NoV) virus like particles (VLPs). Mice were immunized with purified GII.6 NoV VLPs and peptide-bovine serum albumin (BSA) conjugates with the peptide sequence (31 aa) derived from the trypsin cleavage region. An indirect enzyme-linked immunosorbent assay was used to identify positive cell clones during cloning and subcloning, and an in vitro VLP-histo-blood group antigens (HBGAs) binding blockade assay was used to identify mAbs with blocking ability. A total of seven mAbs comprising five (1F7, 1F11, 2B6, 2C4, and 2E10) reactive with major capsid proteins (VP1) and two (1E5 and 2B2) reactive with both VP1 proteins and the peptide were identified. mAb 1F7, 1F11, and 2B6 were identified as blocking antibodies. Sandwich ELISA indicated that all these mAbs recognized soluble GII.6 NoV VLPs. Cross-reactivities with GI.7, GII.3, and GII.4 NoV VLPs were observed in indirect and sandwich ELISA. Western blot analysis indicated that all non-blocking mAbs recognized denatured GII.6 VP1 proteins and blocking mAbs only recognized non-denatured proteins. The in vitro VLP-HBGA binding blockade assay indicated that the three blocking antibodies exhibited blocking effects against GII.6 NoV VLPs, but not GI.7, GII.3, and GII.4 NoV VLPs. Epitope mapping and HBGA blocking assay indicated that mAbs targeting the predicted surface-exposed loop region did not have blocking effects, suggesting a possible important role of this region in regulating NoV-HBGA interactions. This is the first report regarding the characterization of mAbs with blocking ability against GII.6 NoV VLPs. These mAbs might be useful in facilitating our understanding of this group of viruses.

Genipin crosslinked sugar beet pectin-whey protein isolate/bovine serum albumin conjugates with enhanced emulsifying properties

This work aimed to construct conjugates of sugar beet pectin (SBP) with exogenous proteins by adopting a genipin (GP) crosslinking strategy. The conversion processes of two protein, bovine serum albumin (BSA) and whey protein isolate (WPI) to the molecular chains of SBP were investigated using high-performance size-exclusion chromatography technology under varying SBP/exogenous protein weight ratios (SPR) (2:1–20:1) and crosslinking times (1–25 h). When prepared at SPR 10:1, the formed SBP-WPI/BSA-SBP conjugates had notably higher molecular weights of 2151 ± 68 kg/mol and 1758 ± 45 kg/mol, respectively, in comparison with that of SBP (289 ± 16 kg/mol). In terms of reactivity, WPI showed higher reactivity than BSA in crosslinking with SBP by GP. Two representative samples, SBP-WPI-Conjugate and SBP-BSA-Conjugate, prepared at SPR 10:1 after 25 h of crosslinking were further analyzed for surface hydrophobicity (S0) and emulsifying properties. It was found that S0 values of the SBP-WPI-Conjugate and SBP-BSA-Conjugate were significantly lower than that of the control blend samples. Furthermore, the selected conjugates showed superior emulsion stabilizing properties as compared with SBP-exogenous protein blends or individual SBP and exogenous proteins. After storage at 60 °C for 7 d, emulsions stabilized by SBP-WPI-Conjugate and SBP-BSA-Conjugate at concentration of 1 g/100 g emulsion had mean droplet diameters of 0.735 ± 0.023 μm and 0.733 ± 0.011 μm at pH 3.5, and 0.972 ± 0.020 μm and 1.07 ± 0.08 μm at pH 7.0, respectively. Altogether, the work demonstrates that the GP crosslinking method is suitable for upgrading SBP to SBP-protein conjugate with enhanced emulsification properties.

Development of Tripolymeric Triaxial Electrospun Fibrous Matrices for Dual Drug Delivery Applications

Since the first work by Laurencin and colleagues on the development of polymeric electrospinning for biomedical purposes, the use of electrospinning technology has found broad applications in such areas of tissue regeneration and drug delivery. More recently, coaxial electrospinning has emerged as an important technique to develop scaffolds for regenerative engineering incorporated with drug(s). However, the addition of a softer core layer leads to a reduction in mechanical properties. Here, novel robust tripolymeric triaxially electrospun fibrous scaffolds were developed with a polycaprolactone (PCL) (core layer), a 50:50 poly (lactic-co-glycolic acid) (PLGA) (sheath layer) and a gelatin (intermediate layer) with a dual drug delivery capability was developed through modified electrospinning. A sharp increase in elastic modulus after the incorporation of PCL in the core of the triaxial fibers in comparison with uniaxial PLGA (50:50) and coaxial PLGA (50:50) (sheath)-gelatin (core) fibers was observed. Thermal analysis of the fibrous scaffolds revealed an interaction between the core-intermediate and sheath-intermediate layers of the triaxial fibers contributing to the higher tensile modulus. A simultaneous dual release of model small molecule Rhodamine B (RhB) and model protein Fluorescein isothiocynate (FITC) Bovine Serum Albumin (BSA) conjugate incorporated in the sheath and intermediate layers of triaxial fibers was achieved. The tripolymeric, triaxial electrospun systems were seen to be ideal for the support of mesenchymal stem cell growth, as shrinkage of fibers normally found with conventional electrospun systems was minimized. These tripolymeric triaxial electrospun fibers that are biomechanically competent, biocompatible, and capable of dual drug release are designed for regenerative engineering and drug delivery applications.

An Innovative Nanobody-Based Electrochemical Immunosensor Using Decorated Nylon Nanofibers for Point-of-Care Monitoring of Human Exposure to Pyrethroid Insecticides.

A novel ultrasensitive nanobody-based electrochemical immunoassay was prepared for assessing human exposure to pyrethroid insecticides. 3-Phenoxybenzoic acid (3-PBA) is a common human urinary metabolite for numerous pyrethroids, which broadly served as a biomarker for following the human exposure to this pesticide group. The 3-PBA detection was via a direct competition for binding to alkaline phosphatase-embedded nanobodies between free 3-PBA and a 3-PBA-bovine serum albumin conjugate covalently immobilized onto citric acid-decorated nylon nanofibers, which were incorporated on a screen-printed electrode (SPE). Electrochemical impedance spectroscopy (EIS) was utilized to support the advantage of the employment of nanofibrous membranes and the success of the immunosensor assembly. The coupling between the nanofiber and nanobody technologies provided an ultrasensitive and selective immunosensor for 3-PBA detection in the range of 0.8 to 1000 pg mL-1 with a detection limit of 0.64 pg mL-1. Moreover, when the test for 3-PBA was applied to real samples, the established immunosensor proved to be a viable alternative to the conventional methods for 3-PBA detection in human urine even without sample cleanup. It showed excellent properties and stability over time.

Engineering a protease-based and site-specific PEGylation-based strategy for the controlled release of exenatide.

Using the commercially available antidiabetic drug exenatide (exendin-4) as a model peptide, we designed a novel exenatide derivative, termed LEX-1, comprising a 12-mer albumin-binding peptide, a protease-sensitive linker and a native exenatide. In addition, site-specific PEGylation was performed using LEX-1 as a lead sequence to generate four exenatide derivatives (LEX-2 to LEX-5). Moreover, we determined the optimal molecular weight of maleimide-derivatized PEG for the site-specific PEGylation of LEX-1 by an in vitro stability assay and an in vivo hypoglycemic efficacy test. As a result, LEX-3 (PEG10 kDa) exerted enhanced proteolytic stability, rational release rate of free exenatide and the best glucose-stabilizing capability compared with others. In addition, the prolonged hypoglycemic effects of LEX-1 and LEX-3 were demonstrated in type 2 diabetic mice by multiple OGTTs and a hypoglycemic duration test. Furthermore, a pharmacokinetic test was conducted using Sprague Dawley (SD) rats; LEX-3 (PEG10 kDa) showed the best circulating t1/2 of ∼119.7 h for exenatide release from LEX-3, suggesting that LEX-3 has the potential to be developed into a once-weekly antidiabetic agent. The consecutive 8 week treatment of both LEX-1 and LEX-3 exhibited enhanced beneficial efficacies on body weight gain, cumulative food intake, % fat and hemoglobin A1c (HbA1c) reduction compared with exenatide treatment. Meanwhile, the chronic administration of LEX-1 and LEX-3 also effectively improved the blood biochemical indexes. Our results indicate the enhanced antidiabetic effects of LEX-1 and LEX-3, and our strategy of PEGylation and albumin conjugation can be applied to other bioactive agents.

Diphenyltetrazole Modified Bacterial Cellulose Film: Considerations on Heterogeneous Modification and Bioconjugation

Abstract Understanding chemical reactions in bacterial cellulose (BC) films is important to control the insertion of biomolecules that can improve their biomedical properties. Diphenyltetrazole molecules are able to react with tiols under UV irradiation and has been useful for immobilize proteins on different materials. In this work, we show the functionalization of a BC film with diphenyltetrazole and subsequent attempts of conjugation of the bovine serum albumin (BSA) to it. Diphenyltetrazole was synthesized in two steps, and functionalization of it with BC was carried out under ultrasound irradiation. Spectroscopic analysis of the film by UV–Vis and FTIR confirmed the success of the reaction. Attempts to conjugate BSA to the modified cellulose film by photoclick reaction failed under these conditions. Although this methodology is important in the conjugation of biomolecules, we understand that its application in cellulosic materials in heterogeneous phase is disadvantaged due to the half-life of the unstable intermediate and the biomolecule's access to it.

A Nanomaterial-Based Biosensor for the Quantitative Detection of Enrofloxacin Residues in Raw Chicken

HighlightsA nanobiosensor was developed for rapid detection of enrofloxacin residues in chicken meat.5-Sulfosalicylic acid was adopted in a facile method for pretreatment of chicken meat samples.The detection limit of 14.1 µg kg-1 was below the maximum residue limit for chicken meat.The total detection time from sample pretreatment to result report was less than 1.5 h.Abstract. Antibiotic residues in animal-derived food products have been identified as a potential hazard in human health. Hence, a rapid, simple, and cost-effective method for detection of antibiotics in the food supply chain is highly desirable. The objective of this study was to develop a nanomaterial-based biosensor using immunomagnetic beads (IMBs) and quantum dots (QDs) for rapid and sensitive detection of enrofloxacin (ENR) residues in raw chicken. A 5-sulfosalicylic acid-based pretreatment method was adopted to extract ENR from chicken meat and reduce non-specific adsorption caused by complex food matrices. Two sensing elements were designed and fabricated: antibody functionalized IMBs and ENR-bovine serum albumin (BSA) conjugates modified QDs (QDs-BSA-ENR). Target ENR in samples was first captured and separated by IMBs, and then QDs-BSA-ENR, serving as a competitor and detection probe, was used to react with the residual binding sites on the IMB surfaces. With the presence of captured ENR, the binding of QDs-BSA-ENR to IMBs was competitively inhibited. Finally, the fluorescence intensity of reporting QDs in the QDs-BSA-ENR-IMBs complex at a wavelength of 614 nm was measured for the quantitation of target antibiotics. Under the optimum conditions, the proposed method allowed sensitive detection of ENR in a linear range from 1 to 100 ng mL-1 with a limit of detection (LOD) of 0.94 ng mL-1. The LOD for spiked chicken meat was 14.1 µg kg-1, which was below the maximum residue limits (MRLs) regulated in China and the European Union. The whole analytical procedure from food sampling to result report could be finished in less than 1.5 h. This nanobiosensor showed high potential for rapid and low-cost detection of ENR residues in the poultry supply chain to enhance food safety. Keywords: Enrofloxacin, Immunomagnetic beads, Nanobiosensor, Poultry, Quantum dots, Rapid detection.

Simultaneous detection of plant growth regulators jasmonic acid and methyl jasmonate in plant samples by a monoclonal antibody-based ELISA.

Methyl jasmonate (MeJA) and its free-acid form, jasmonic acid (JA), collectively referred to as jasmonates (JAs), are natural plant growth regulators that are widely present in higher plants. Simultaneous detection of JA and MeJA in plant samples is of significance and is a great challenging issue. In this study, coupling with two extraction methods, a sensitive monoclonal antibody (mAb) based enzyme-linked immunosorbent assay (ELISA) for simultaneous detection of JA and MeJA in plant samples was developed. The JA-bovine serum albumin (BSA) conjugate was used as an immunogen for the production of mAb. As the produced mAb exhibited higher recognition ability towards MeJA than towards JA, ELISA was established using MeJA as the standard. Under optimal experimental conditions, the IC50 and LOD values of ELISA for MeJA were 2.02 ng mL-1 and 0.20 ng mL-1, respectively. In the first extraction method, MeJA in plant samples was evaporated and only JA was extracted. In the second extraction method, both JA and MeJA were extracted. After methylation, JA in the extracts was converted into MeJA, and the whole MeJA in the extracts was measured by ELISA. Plant samples including the leaves of Salvia splendens, the flowers of Salvia splendens and the fruit of grapes were collected. JA and MeJA in these samples were detected by the proposed ELISA. It was found that the concentrations of JA in these three plant samples were about 3-5 times higher than those of MeJA in those samples. ELISA was also confirmed by HPLC. There was a good correlation between ELISA and HPLC.

Rapid detection of 21 β-lactams using an immunochromatographic assay based on the mutant BlaR-CTD protein from Bacillus Licheniformis.

In this study, a gold immunochromatographic assay (GICA) based on a penicillin receptor protein (PBP) is proposed to simultaneously detect penicillin, cephalosporin, and carbapenem antibiotics in milk and chicken. A mutant PBP, carboxy-terminal of the β-lactam sensor-transducer protein (BlaR-CTD, I188KS19CG24C) from Bacillus licheniformis ATCC14580 was expressed in E. coli as a soluble His-tag fusion protein. The purified BlaR-CTD mutant was labeled with colloidal gold (CG-BlaR-CTD-M) and applied to develop GICA. The GICA method was performed based on a competitive form, where the binding of the ampicillin-bovine serum albumin conjugate (Amp-BSA) to CG-BlaR-CTD-M was inhibited by free β-lactams in samples. The performance of the assay was confirmed by analysis of milk and chicken samples. The limits of detection of 21 β-lactam antibiotics met the maximum residue limits set by the European Union and China. The GICA gave results within 10 min, thus providing a rapid and reliable method for the on-site detection of multiple β-lactams in milk and chicken samples.

A Self-Assembled "Albumin-Conjugate" Nanoprobe for Near Infrared Optical Imaging of Subcutaneous and Metastatic Tumors.

The need for in situ accurate identification of tumor assisted real-time image-guided surgical resection calls for new near-infrared fluorescence agents with high tumor-sensitivity and excellent biocompatibility. Here, an albumin-conjugate nanoparticle system HSA-Er-RI-Cl was designed, synthesized, and applied in cancer imaging, which simultaneously achieved the EPR effect, hypoxia-targeting, and EGFR-targeting property. Our novel nanoprobe is composed of human serum albumin (HSA) and double-targeting small molecule conjugate (Er-RI-Cl): a hypoxia-targeting heptamethine carbocyanine dye (RI-Cl) conjugated with a clinic anti-EGFR antagonist (Erlotinib) by covalent bonding. This conjugate could bind to albumin proteins, forming albumin-conjugate complexes, and those complexes self-assemble into particles with diameters of approximately 100 nm in the aqueous solution. The tumor hypoxia and EGFR targeting specificity of HSA-Er-RI-Cl was, respectively, evaluated in vitro and in vivo. Using murine xenograft subcutaneous and brain metastatic tumor models, we demonstrated that HSA-Er-RI-Cl is a highly potent tumor-targeting NIR agent for noninvasive imaging with remarkable tumor localization and excellent pharmacokinetic properties.

Aflatoxin levels in maize and peanut and blood in women and children: The case of Timor-Leste

Aflatoxins are toxic fungal metabolites produced by Aspergillus sp. with carcinogenic properties that are a common food contaminant of many crops including maize and peanuts. In Timor-Leste malnutrition and children’s stunting are frequent and maize and peanuts are staple foods. This study aimed to provide information on aflatoxin exposure nationally. The study measured levels of aflatoxin in locally-produced maize and peanuts (296 samples) and of aflatoxin-albumin conjugate in blood samples of women and young children (514 and 620 respectively) across all municipalities. The average concentration of aflatoxin in the grain samples was low with most maize (88%) and peanut (92%) samples - lower than European Commission tolerated aflatoxin level. Although aflatoxin–albumin conjugate was detected in more than 80% of blood samples, the average concentration in children and adults of 0.64 and 0.98 pg mg−1 alb, respectively, is much lower than in other similar rural-based countries. Although low in concentration, blood aflatoxin levels and aflatoxin contamination levels in maize across municipalities were correlated significantly for mothers (R2 = 37%, n = 495) but not for children (R2 = 10%). It is unlikely that the consumption of aflatoxin contaminated grain is a causative factor in the current level of malnutrition and stunting affecting Timor-Leste children.

Bovine Serum Albumin Amplified Reactive Oxygen Species Generation from Anthrarufin-Derived Carbon Dot and Concomitant Nanoassembly for Combination Antibiotic–Photodynamic Therapy Application

Amplification of reactive oxygen species (ROS) generation through covalent conjugation of bovine serum albumin (BSA) with newly synthesized, ROS-producing carbon dots (CDs) upon visible light irradiation is reported for the first time. Derivatization of surface carboxyl functional groups of Anthrarufin-derived, green-emitting CD with the amine functionality of BSA ushers distinct changes in the photophysics of CD including an unprecedented ∼50 nm shift in its excitation maxima, decrease in fluorescence lifetime, and concomitant increase in ROS generation. Substantial conformational changes of BSA were witnessed upon conjugation with CD, rendering the BSA-CD conjugate resistant to pepsinolysis. A protease-proof nanoassembly was derived from the BSA-CD conjugate through desolvation that simultaneously hosts a prototype antibiotic and generates ROS with excellent efficiency, making it an attractive platform for antibacterial photodynamic therapy (A-PDT) applications. Systemic annihilation of both Gram-positive and -negative bacteria was achieved with the BSA-CD nanoassembly and envisioned as alternatives to traditional photosensitizers.

A surface plasmon resonance immunoassay for the rapid analysis of methamphetamine in forensic oral fluid.

BackgroundCurrent chromatographic methods applied for the forensic analysis of methamphetamine are costly, time‐consuming, and require complicated pretreatment procedures. Thus, the rapid detection of methamphetamine is a critical and unmet need. In this study, a surface plasmon resonance (SPR) system based on indirect inhibitive immunoassay was designed for the analysis of methamphetamine in forensic oral fluid samples.MethodsFor the inhibition immunoassay, the diluted oral fluid was mixed with methamphetamine antibody and then injected into the SPR sensor chip. The biosensor chip was constructed by covalently immobilizing of methamphetamine‐bovine serum albumin conjugate onto a carboxymethyl dextran surface at an optimized pH. The concentration of antibody was also optimized.ResultsThe SPR biosensor showed good sensitivity with a limit of detection of 0.44 ng/mL and was comparable or lower than the pre‐existing methods. The method was finally tested using oral fluid samples from 20 suspected drug abusers in forensic cases, and it provided an acceptable recovery of 113.2%, indicating good anti‐interference capability of the SPR sensor.ConclusionThe SPR biosensor was rapid, reproducible, and had a great potential approach for the forensic detection of methamphetamine.

A competitive thrombin-linked aptamer assay for small molecule: aflatoxin B1.

We described a competitive thrombin-linked aptamer assay for small molecule, using aflatoxin B1 (AFB1) as a model, taking advantage of aptamer affinity binding and enzymatic activity of thrombin. We designed a dual functional DNA probe that contained the aptamer sequence for thrombin and the aptamer sequence for AFB1. Thrombin was labeled on the DNA probe by affinity binding between thrombin and anti-thrombin aptamer. This thrombin-labeled DNA probe was attached on AFB1-bovine serum albumin conjugate (BSA-AFB1) coated on a microplate through the affinity interaction between AFB1 and anti-AFB1 aptamer. The thrombin attached on the microplate catalyzed the cleavage of peptide substrate into detectable product, generating signal for detection. In the presence of AFB1, free AFB1 competed with BSA-AFB1 in the binding to the limited amount of DNA probe, leading to signal decrease. Detection of AFB1 was achieved by measuring the signal change. Under optimized conditions, AFB1 was successfully detected in the range from 0.5nM to 1μM when fluorogenic peptide substrate of thrombin and fluorescence analysis were applied. The use of chromogenic peptide substrate in the assay allowed the detection of AFB1 ranging from 0.5 to 125nM by simple absorbance analysis. The thrombin-linked aptamer assay showed good selectivity towards AFB1, and enabled the detection of AFB1 spiked in diluted beer and corn flour. This TLAA strategy extends the analytical application of thrombin and aptamers in detection of small molecules. Graphical abstract.

Glucocorticoid receptor involvement in goose (Anas cygnoides) pituitary somatotroph differentiation induced by glucocorticoids during embryonic development

ABSTRACT 1. In this study, geese (Anas cygnoides) embryonic pituitary cells were cultured in vitro to determine if glucocorticoids could induce growth hormone (GH) expression and to investigate the molecular mechanisms involved in this process. 2. On embryonic day 15 (e15) and e20 the pituitary cells were treated with corticosterone (CORT), membrane impermeable bovine serum albumin–conjugate corticosterone (CORT-BSA), dexamethasone (DEX), and a glucocorticoid receptor (GR) antagonist (RU486) to detect responsiveness of somatotrophs to glucocorticoids. 3. Treatment with CORT, CORT-BSA, and DEX for as little as 6 h increased the percentage of GH-positive cells (P < 0.01) and increased GH mRNA expression (P < 0.01) in e15 goose pituitary cells compared to the control. CORT significantly increased the level of GH protein secreted from cultured e15 goose embryonic pituitary cells, and CORT-BSA increased GH secretion from e20 goose embryonic pituitary cells. 4. A significant increase was observed in the glucocorticoid receptor in GR transcription levels (P < 0.01) with CORT, CORT-BSA, and DEX treatment. Furthermore, the CORT-stimulated GH mRNA expression was completely negated by pre-treatment with RU486. 5. These findings demonstrate that glucocorticoids can stimulate somatotroph differentiation in vitro, as characterised by enhanced GH protein secretion andmRNA expression in cultured geese embryonic pituitary cells. The membrane GR was involved in pituitary somatotroph differentiation induced by glucocorticoids during the embryonic development of geese.

Development of a progesterone-specific IgE assay for diagnosing patients with suspected progestogen hypersensitivity

BackgroundProgesterone hypersensitivity (PH) manifests as a spectrum of allergic symptoms during the luteal phase of the menstrual cycle. Confirming progesterone-specific immunoglobulin E (IgE; sIgE) by skin testing is unreliable because of irritant responses. ObjectiveTo develop a progesterone sIgE assay to assist in diagnosing PH. MethodsA progesterone–bovine serum albumin (BSA) conjugate was characterized and used to analyze sera collected from women in our center with suspected PH in a 1-batch enzyme-linked immunosorbent assay (ELISA) to establish high, low and negative cut-points. Sera collected from healthy nonatopic female subjects and from women with classical PH symptoms were included as negative and positive controls, respectively. Values exceeding the average negative control (OD + 3× the standard deviation) were considered positive. These cut-points were subsequently used to establish positive and negative results for serum from women with suspected PH received from other centers. A subset of high positive sera was used for ELISA-inhibition and in a beta-hexosaminidase mediator release assay to evaluate the specificity and functional relevance of progesterone-specific serum IgE, respectively. The numbers of true negative, false negative, true positive, and false positive samples were determined. ResultsThe direct progesterone sIgE ELISA results ranged from high positive to low positive and negative compared with healthy nonatopic control sera. Enzyme-linked immunosorbent assay inhibition and beta-hexosaminidase mediator release confirmed specificity and functional relevance of progesterone-sIgE, respectively. The sensitivity, specificity positive predictive values and negative predictive values were found to be 82%, 100%, 86%, and 100%, respectively, using the mediator release assay results as the gold standard. ConclusionThis assay has a good specificity and positive predictive value for screening women with suspected PH for progesterone sIgE.

Label-free gold nanoclusters as quenchable fluorescent probes for sensing olaquindox assisted by glucose oxidase-triggered Fenton reaction

ABSTRACTGlucose oxidase (GOx) catalyses oxidation of glucose accompanied with the generation of hydrogen peroxide. With the addition of Fe2+, hydroxyl radical produced by Fenton reaction between hydrogen peroxide and Fe2+ may quench the fluorescence of gold nanoclusters. In this work, a fluorescent enzyme-linked immunosorbent assay with gold nanoclusters was designed with a straightforward signal output, in which the fluorescence of gold nanoclusters was quenched by GOx-triggered Fenton reaction. Olaquindox was selected as a target analyte. Gold nanoclusters capped with bovine serum albumin and GOx-linked olaquindox conjugates were successfully prepared. Olaquindox in samples directly competed with the GOx-linked olaquindox conjugates for binding immobilized antibody. Consequently, the fluorescence signal increased with the amount of olaquindox. Under optimal conditions, the fluorescent enzyme-linked immunosorbent assay exhibited a favorable performance to detect olaquindox in swine feeds, demonstrating a good linear range from 1.0 µg kg−1 to 150 µg kg−1 with a reliable correlation coefficient (R2 = 0.9918); the limit of detection was 0.68 µg kg−1. Average recoveries in spiked samples were 85.3% to 113.5%. The proposed strategy is a promising approach for the detection of olaquindox and other harmful small molecules.

Albumin Based Nanoparticles for Detection of Pancreatic Cancer Cells.

Molecular imaging of cancer cells using effective drug targeting systems are most interested research area in recent years. Albumin protein is a soluble and most abundant protein in circulatory system. It has a ligand-binding function and acts as a transport protein. Researchers are interested in developing albumin based nanostructured specific anti-tumor drugs in cancer therapy. Pancreatic cancer treatment or drug design for targeted pancreatic cancer cell has great importance due to it has a high mortality rate comparing other cancer types. In this article, our goal is to develop new targeting nanoparticles based on the conjugation of albumin and Hyaluronic Acid (HA) for pancreatic cancer cells. In this article, we proposed a new technique for conjugation of albumin (BSA) and HA in nano formation. Firstly, cationic BSA is synthesized. Then, BSA-HA conjugation is obtained by interacted cationic BSA with 1000 ppm HA. Secondly, nano BSA-HA particles and nano BSA particles were synthesized according to AmiNoAcid Decorated and Light Underpinning Conjugation Approach (ANADOLUCA) method which provides a special cross-linking strategy for biomolecules using ruthenium-based amino acid monomer haptens. After characterization studies, in vitro cytotoxic activity of synthesized nano BSA-HA particles were determined for PANC-1 ATCC® CRL146 cells. According to the data, nano BSA and nano BSA-HA particles synthesized uniquely using special ruthenium-based amino acid decorated cross-linking agent, (MATyr)2-Ru-(MATyr)2.based on ANDOLUCA method. Characterization results showed that there was not any change in protein folding structures during nano formation process. In addition, nano protein particles gained fluorescence feature. When interacting synthesized nano BSA and nano BSA-HA particles with pancreatic cells, it was found that BSA nanoparticles were usually around cells and membranes, but BSA-HA nanoparticles were identified around the cells, in the cytoplasm inside the cell, and next to the cell nucleus. So, nano BSA-HA particles could be used as cancer cell imaging agent for PANC-1 ATCC® CRL146 cells. The satisfactory conclusion of this study is that synthesized nano BSA-HA particles are fundamental materials for targeting pancreatic cancer cells due to HA receptors located on pancreatic cancer cells and imaging agents due to fluorescence feature of the BSA-HA nanoparticles.

Development of a Novel Imaging Agent for Determining Albumin Uptake in Solid Tumors.

The purpose of this study was to investigate the albumin-binding compound 111In-C4-DTPA as an imaging agent for the detection of endogenous albumin accumulation in tumors. 111In-C4-DTPA was injected in healthy nude mice for pharmacokinetic and biodistribution studies (10min, 1, 6, 24, and 48h, n = 4) and subsequently in tumor-bearing mice for single-photon emission computed tomography/X-ray-computed tomography (SPECT/CT) imaging studies. Four different human tumor xenograft models (LXFL529, OVXF899, MAXFTN401, and CXF2081) were implanted subcutaneously unilaterally or bilaterally (n = 4-8). After intravenous administration of 111In-C4-DTPA, SPECT/CT images were collected over 72h at 4-6 time points. Additionally, gamma counting was performed for the blood, plasma, lungs, heart, liver, spleen, kidneys, muscle, and tumors at 72h post-injection. 111In-C4-DTPA bound rapidly to circulating albumin upon injection, and the radiolabeled albumin conjugate thus formed was stable in murine and human serum. SPECT/CT images demonstrated a time-dependent uptake with a maximum of 2.7-3.8%ID/cm3 in the tumors at approximately 24h post-injection and mean tumor/muscle ratios in the range of 3.2-6.2 between 24 and 72h post-injection. The kidneys and bladder were the predominant elimination organs. Gamma counting at 72h post-injection showed 1.3-2.5%ID/g in the tumors and mean tumor/muscle ratios in the range of 4.9-9.4. 111In-C4-DTPA bound rapidly to circulating albumin upon injection and showed time-dependent uptake in the tumors demonstrating a potential for clinical application as a companion imaging diagnostic for albumin-binding anticancer drugs.