Design of single-domain VHH antibodies to increase the binding activity in SPR amine coupling.

Single-domain antibodies, or VHH, nanobodies, are attractive tools in biotechnology and pharmaceuticals due to their favorable biophysical properties. Single-domain antibodies have potential for use in sensing materials to detect antigens, and in this paper, we propose a generic design strategy of single-domain antibodies for the highly efficient use of immobilized antibodies on a sensing substrate. Amine coupling was used to immobilize the single-domain antibodies on the substrate through a robust covalent bond. First, for two model single-domain antibodies with lysines at four highly conserved positions (K48, K72, K84, and K95), we mutated the lysines to alanine and measured the binding activity of the mutants (the percentage of immobilized antibodies that can bind antigen) using surface plasmon resonance. The two model single-domain antibodies tended to have higher binding activities when K72, which is close to the antigen binding site, was mutated. Adding a Lys-tag to the C-terminus of single-domain antibodies also increased the binding activity. We also mutated the lysine for another model single-domain antibodies with the lysine in a different position than the four residues mentioned above and measured the binding activity. Thus, single-domain antibodies immobilized in an orientation accessible to the antigen tended to have a high binding activity, provided that the physical properties of the single-domain antibodies themselves (affinity and structural stability) were not significantly reduced. Specifically, the design strategy of single-domain antibodies with high binding activity included mutating the lysine at or near the antigen binding site, adding a Lys-tag to the C-terminus, and mutating a residue away from the antigen binding site to lysine. It is noteworthy that mutating K72 close to the antigen binding site was more effective in increasing the binding activity than Lys-tag addition, and immobilization at the N-terminus close to the antigen binding site did not have such a negative effect on the binding activity compared to immobilization at the K72.

Imaging-Guided Chemo-Photothermal Polydopamine Carbon Dots for EpCAM-Targeted Delivery toward Liver Tumor.

We demonstrate a versatile nanoparticle with imaging-guided chemo-photothermal synergistic therapy and EpCAM-targeted delivery of liver tumor cells. EpCAM antibody (anti-EpCAM) and Pt(IV) were grafted onto the polydopamine carbon dots (PDA-CDs) by the amidation reaction. The EpCAM antibody of particles enables the targeted interaction with liver progenitor cells due to their overexpressed EpCAM protein. The tetravalent platinum prodrug [Pt(IV)] induces apoptosis with minimum toxic side effects through the interaction between cisplatin and tumor cell DNA. The nanoparticles displayed stable photothermal property and considerable anti-tumor therapeutic effect in vivo. Coupling with cellular imaging due to their fluorescence property, anti-EpCAM@PDA-CDs@Pt(IV) offers a convenient and effective platform for imaging-guided chemo-photothermal synergistic therapy toward liver cancers in the near future.

Covalently Immobilized Regenerable Immunoaffinity Layer with Orientation-Controlled Antibodies Based on Z-Domain Autodisplay.

A regenerable immunoaffinity layer comprising covalently immobilized orientation-controlled antibodies was developed for use in a surface plasmon resonance (SPR) biosensor. For antibody orientation control, antibody-binding Z-domain-autodisplaying Escherichia coli (E. coli) cells and their outer membrane (OM) were utilized, and a disuccinimidyl crosslinker was employed for covalent antibody binding. To fabricate the regenerable immunoaffinity layer, capture antibodies were bound to autodisplayed Z-domains, and then treated with the crosslinker for chemical fixation to the Z-domains. Various crosslinkers, namely disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS) and poly (ethylene glycol)-ylated bis (sulfosuccinimidyl)suberate (BS(PEG)5), were evaluated, and DSS at a concentration of 500 µM was confirmed to be optimal. The E. coli-cell-based regenerable HRP immunoassay was evaluated employing three sequential HRP treatment and regeneration steps. Then, the Oms of E. coli cells were isolated and layered on a microplate and regenerable OM-based HRP immunoassaying was evaluated. Five HRP immunoassays with four regeneration steps were found to be feasible. This regenerable, covalently immobilized, orientation-controlled OM-based immunoaffinity layer was applied to an SPR biosensor, which was capable of quantifying C-reactive protein (CRP). Five regeneration cycles were repeated using the demonstrated immunoaffinity layer with a signal difference of <10%.

Design and site-directed immobilization of single-chain Fv antibody to polystyrene latex beads via material-binding peptides and application to latex turbidimetric assay.

In this study, immobilization of single-chain Fv (scFv) antibodies on the surfaces of polystyrene (PS) latex beads via material-binding peptides was investigated for sensitive immuno-turbidimetric assay of C-reactive protein (CRP). Anti-CRP scFvs fused with polystyrene-binding peptide (PS-tag) and poly(methylmethacrylate)-binding peptide (PMMA-tag) were over-expressed in Escherichia coli cells and recovered in the active form following refolding. The beads with PMMA-tag-fused scFv (scFv-PM) were successfully suspended with sufficient dispersion at pH 8.0. Three types of alternative scFv-PMs with a penta-asparatic acid tag (D5-tag) introduced at different positions were then designed. All of the D5-tagged scFv-PMs were successfully immobilized on the surfaces of beads with no significant change in the diameter of the latex beads at pH levels ranging from 6.0 to 8.0. According to the results of turbidimetric assay for the detection of CRP, 13 ng/ml of CRP was detectable using beads with D5-tagged scFv-PMs at 400 ng/cm3, and no turbidity change was observed in the absence of antigen. When the density of scFv-PM was 250 ng/cm2, which was 63% of the maximum density, the beads were dispersed well and reactive with the antigen at a concentration range comparable to those with D5-tagged scFv-PMs. These results indicate that controlling charge density on the surface of beads after site-directed immobilization is definitely important in order to maintain high levels of dispersion and reactivity. Thus, the usefulness of the scFv-PM as well as D5-tagged scFv-PMs developed in the present study should be significant when used as ligand antibodies in the preparation of immuno-latex beads.

Development of a monoclonal antibody-based immunochromatographic assay for the detection of carbamazepine and carbamazepine-10, 11-epoxide.

In this study, haptens were designed to produce highly sensitive and specific monoclonal antibodies (mAb) against carbamazepine (CBZ) and its metabolite carbamazepine-10, 11-epoxide (CBZ-EP). According to the results of our competitive enzyme-linked immunosorbent assay (ic-ELISA), the half-maximum inhibitory concentration values for anti-CBZ and anti-CBZ-EP mAb were 0.18 and 0.59 ng/mL, respectively. An immunochromatographic assay (ICA) was developed for the determination of CBZ and CBZ-EP concentrations. This method can provide visible limits of detection ranging from 0.25 to 1 ng/mL, and cut-off limits ranging from 5 to 10 ng/mL, and takes 10 min to evaluate with the naked eye. Importantly, these observations were consistent with those obtained by ic-ELISA and liquid chromatography-mass spectrometry. The ICA assay represented a reliable, fast, and high-throughput method for the determination of CBZ and CBZ-EP in serum samples.

Sensitive detection of carcinoembryonic antigen (CEA) by a sandwich-type electrochemical immunosensor using MOF-Ce@HA/Ag-HRP-Ab2 as a nanoprobe.

Sandwich-type electrochemical immunosensor was one of the main methods for detecting carcinoembryonic antigen (CEA). In this work, using Ce-MoF as the skeleton precursor, hyaluronic acid (HA) was coated on the surface of Ce-metal organic framework (Ce-MoF), which loaded with silver nanoparticles (Ag NPs) and horseradish peroxidase (HRP) to catalyze H2O2 and double amplified the current signal. Thus, a sensitive sandwich-type electrochemical immunosensor (Ce-MoF@ HA/Ag-HRP) was designed to detect carcinoembryonic antigen (CEA). The designed immunosensor used Au NPs to enhance the ability of attach more the first antibody (Ab1). This was due to Au NPs had good electrical conductivity and biocompatibility to accelerate electron transfer on the surface of the electrode. HA was riched in -COOH, -OH and had excellent biocompatibility, which can carry more Ag NPs to catalyze H2O2. Finally, the prepared sandwich-type electrochemical immunosensor had excellent biocompatibility and great catalytic performance. The immunosensor can be tested within 30 min and the logarithm of the current signal and CEA concentration showed a broad linear response range of 1 pg ml-1-80 ng ml-1, and the detection limit of CEA was 0.2 pg ml-1. More importantly, the proposed immunosensor had good reproducibility, selectivity, stability and without matrix effect. This confirmed that the proposed immunosensor had broad prospects in early clinical trials.

Rapid Detection of Staphylococcal Enterotoxin-B by Lateral Flow Assay.

A cohort of monoclonal antibodies (mAbs) were generated against Staphylococcal enterotoxin-B (SEB) and selected by double sandwich enzyme-linked immunosorbent assay (ELISA) for solution capture of the toxin. Clonal hybridoma cell lines were established and a pair of anti-SEB mAbs selected for the development of a sandwich ELISA. Immobilized 3D6 mAb (IgG1, kappa) when paired with 4C9 mAb (IgG1, kappa) conjugated to horseradish peroxidase generates a typical dose-response curve with an EC50 of 24.8 ng/mL for purified SEB using chemiluminescent detection. These mAbs bind SEB by Western blot and ELISA binding to classical enterotoxin serotypes show that the 3D6 mAb binds both SEB and the SEC1 serotypes, whereas 4C9 binds only SEB. These mAbs effectively port onto lateral flow test strips with a visual detection sensitivity for SEB of 5 ng/mL in <10 minutes using a 4C9 conjugated to a 40 nm gold reporter.

Enhancing chimeric hydrophobin II-vascular endothelial growth factor A165 expression in Pichia pastoris and its efficient purification using hydrophobin counterpart.

We report cloning and expressing of recombinant human VEGF-A165, fused at the N-terminal with Hydrophobin II (HFBII) from Trichoderma reseei, in yeast Pichia pastoris. We validated the construct using SDS-PAGE and ELISA against VEGF-A165 and efficiently performed protein purification and enrichment based on HFBII counterpart and using an aqueous two-phase system (ATPS) with nonionic surfactant X-114. We studied the effects of various culture medium additives and interaction effects of positive factors to increase the recombinant HFBII-VEGF-A165 production. Supplementing the Pichia pastoris cell culture medium with Mg2+, Polysorbate 20 (PS 20), and 4-phenylbutyrate (PBA) improved the expression of the chimeric protein. Orthogonal experiments showed that the optimal condition to achieve maximal HFBII-VEGF-A165 production was with the addition of PBA, PS 20, and MgSO4. Under this condition, the production of the target protein was 4.5 times more than that in the medium without the additives. Overall, our approach to produce chimeric HFBII-VEGF-A165 and selectively capture it in ATPS is promising for large-scale protein production without laborious downstream processing.

Determination of Real Time in Vivo Drug Receptor Occupancy for a Covalent Binding Drug as a Clinical Pharmacodynamic Biomarker by Immunocapture-LC-MS/MS.

We report a novel immunocapture (IC)-LC-MS/MS methodology to directly measure real time in vivo receptor occupancy (RO) for a covalent binding drug in blood lysate. A small molecule quencher was added immediately after sample collection to convert the free receptor to a quencher-bound receptor (QB-R) which was measured with the drug-bound receptor (DB-R) simultaneously by LC-MS/MS after immunocapture enrichment, followed by trypsin digestion. Addition of the quencher is necessary to prevent the free receptor from ex vivo binding with the drug. The real time RO was calculated based on the concentrations of DB-R and the free receptor (which is now QB-R) that were obtained from each sample. This strategy has been successfully applied to the measurement of the RO for Bruton's tyrosine kinase (BTK) in the blood lysate of monkeys after dosing with branebrutinib (BMS-986195), a covalent BTK inhibitor being evaluated to treat rheumatoid arthritis. A custom-made quencher, which is more reactive to BTK than branebrutinib, was added in excess amount to bind with all available free BTK to form quencher-bound BTK (QB-BTK) during blood sample collection. To measure a wide range of % BTK RO, including those of <5% or >95%, the required LLOQ at 0.125 nM for QB-BTK and 0.250 nM for drug-bound BTK (DB-BTK) in blood lysate were successfully achieved by using this IC-LC-MS/MS strategy. This proof-of-concept assay demonstrated its suitability with high throughput for real time in vivo BTK RO measurement as a pharmacodynamic (PD) biomarker for clinical drug development.

Fluorescent analysis of Staphylococcus aureus by using daptomycin and immunoglobulin G for dual sites affinity.

A dual sites affinity protocol was developed for fluorescent analysis of Staphylococcus aureus (S. aureus) by employing daptomycin and immunoglobulin G (IgG) as the recognition elements. Pig IgG immobilized on microplate was employed as the first recognition element to capture S. aureus owing to the fact that the Fc segment of mammal IgG can selectively bind with protein A on the surface of the target bacteria. Meanwhile, fluorescein isothiocyanate-conjugated daptomycin was employed as the second recognition element as well as the signal tracer for the target bacteria utilizing the binding capability of daptomycin to Gram-positive bacteria. S. aureus can be analyzed within a concentration range of 5.0 × 103-5.0 × 108 CFU mL-1 with a detection limit of 3.6 × 103 CFU mL-1. The analytical process can be accomplished within 1.5 h by using a pre-coated microplate. The dual sites affinity protocol can exclude the interference led by Gram-negative bacteria and other common Gram-positive bacteria. We have successfully applied it to analyze S. aureus in spiked lake water and physiological saline injection samples, and the recovery values ranged from 88.0% to 120.0%. The results demonstrate its application potential for environmental sanitation and drug safety control.

Immunoaffinity microcryogels for purification of transferrin.

Immunoaffinity chromatography has a huge interest in the biomedical and biotechnological fields, in particular for one-step isolation, purification and removal of analyte compounds. In this study, uniform-sized microcryogels, a new type of cryogels, were synthesized using 2-hydroxyetyhl methacrylate and epoxy-group-containing monomer, glycidyl methacrylate for purification of a plasma protein, transferrin. Immunoaffinity microcryogels containing anti-Tf antibodies were characterized by Raman spectroscopy, Fourier transform infrared spectroscopy, optical microscopy, scanning electron microscopy, density measurements and swelling tests. Adsorption studies in aqueous media were carried out in order to examine the effects of medium pH, initial concentration of analyte and contact time. It was found that the optimum pH was 6.0 and the maximum adsorption capacity of immunoaffinity microcryogels at this pH value found to be 9.82 mg/g. The KL constant for Langmuir isotherm was calculated as 2.65 mL/mg. The maximum adsorption capacity obtained from experimental studies is also very close to the calculated Langmuir adsorption capacity (11.27 mg/g). Langmuir adsorption isotherms and pseudo-second-order kinetic models are consistent with the adsorption process, which means that the adsorption is single layered and chemically controlled. The purity of the eluted hsTf from plasma was about 84% with yield about 82%. After the tenth use of the same microcryogels, the maximum hsTf adsorption capacity decreased by about 20%. The results indicated that the immunoaffinity microcryogels having anti-Tf antibody ligands could be a safe and cost-friendly method for purification of transferrin.

Modulating the antibody density changes the uptake and transport at the blood-brain barrier of both transferrin receptor-targeted gold nanoparticles and liposomal cargo.

Transport of the majority of therapeutic molecules to the brain is precluded by the presence of the blood-brain barrier (BBB) rendering efficient treatment of many neurological disorders impossible. This BBB, nonetheless, may be circumvented by targeting receptors and transport proteins expressed on the luminal surface of the brain capillary endothelial cells (BCECs). The transferrin receptor (TfR) has remained a popular target since its original description for this purpose, although clinical progression of TfR-targeted drug constructs or nanomedicines remains unsuccessful. One proposed issue pertaining to the use of TfR-targeting in nanomedicines is the efficient tuning of the ligand density on the nanoparticle surface. We studied the impact of TfR antibody density on the uptake and transport of nanoparticles into the brain, taking a parallel approach to investigate the impact on both antibody-functionalized gold nanoparticles (AuNPs) and cargo-loaded liposomes. We report that among three different low-range mean ligand densities (0.15, 0.3, and 0.6 ∗ 103 antibodies/µm2), the highest density yielded the highest ability towards both targeting of the BCECs and subsequent transport across the BBB in vivo, and in vitro using primary cultures of the murine BBB. We also find that TfR-targeting on liposomes in the mouse may induce severe adverse effects after intravenous administration.

Microfluidic based impedance biosensor for pathogens detection in food products.

A MEMS-based impedance biosensor was designed, fabricated, and tested to effectively detect the presence of bacterial cells including E. coli O157:H7 and Salmonella typhimurium in raw chicken products using detection region made of multiple interdigitated electrode arrays. A positive dielectrophoresis based focusing electrode was used in order to focus and concentrate the bacterial cells at the centerline of the fluidic microchannel and direct them toward the detection microchannel. The biosensor was fabricated using surface micromachining technology on a glass substrate. The results demonstrate that the device can detect Salmonella with concentrations as low as 10 cells/mL in less than 1 h. The device sensitivity was improved by the addition of the focusing electrodes, which increased the signal response by a factor between 6 and 18 times higher than without the use of the focusing electrodes. The biosensor is selective and can detect other types of pathogen by changing the type of the antibody immobilized on the detection electrodes. The device was able to differentiate live from dead bacteria.

Synthesis of multifunctional fluorescent magnetic nanoparticles for the detection of Alicyclobacillus spp. in apple juice.

An approach based on multifunctional fluorescent magnetic nanoparticles was proposed for the enrichment and identification of Alicyclobacillus spp. in apple juice simultaneously. The prepared Fe3O4 magnetic particles (MNPs) were modified by the sol-gel process and a silica shell was formed to improve the reactivity, and then the obtained MNPs@SiO2-SH nanoparticles were conjugated with Thioglycolic acid functionalized CdTe/CdS QDs via thiols chemistry. The characteristic evaluation results indicated that the MNPs-QD nanocomposites exhibited good magnetic properties and optical characterization. The polyclonal anti-Alicyclobacillus IgG antibody was immobilized onto the surface of MNPs-QD materials via esterification reactions. The maximum antibody immobilization capacity was 119.62 µg/mg and the adsorption reaction could be accomplished in 60 min. The adsorption process could be represented by Langmuir model and pseudo-second order kinetics equation, respectively. Based on the high immunocapture efficiency and sensitive fluorescence characteristics, the obtained MNPs-QDs-antibody conjugates could be applied to recognize the contamination of Alicyclobacillus spp. and a quantitative analysis method was established for target cells detection. The minimum quantitative limit was 104 CFU (colony forming unit)/mL and the testing process could be completed in 90 min. The results indicated that the MNPs-QDs-antibody conjugates can be successfully applied for immunocapture and detection of Alicyclobacillus spp. in apple juice. That is to say, the developed MNPs-QDs-antibody conjugates have exhibit more attractive and great potential for the immunocapture and recognition of target bacteria, fully demonstrated a new method for enrichment and rapid detection of Alicyclobacillus spp. in fruit juices.

Electrochemical immunosensor based on chitosan/conductive carbon black composite modified disposable ITO electrode: An analytical platform for p53 detection.

In this study, we fabricated a label-free electrochemical immunosensor for sensitive and selective detection of tumor marker p53. This immunosensor was based on chitosan/carbon black composite (Chitosan-CB) layer coated ITO electrode. This composite was utilized for enhancement of the conductivity of the immunosensor. Anti-p53 antibodies were captured on the modified ITO electrode through the cross-linking of chitosan and glutaraldehyde. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques were utilized for electrochemical characterization of the proposed immunosensor. Moreover, the biosensor construction steps were monitored by using scanning electron microscopy (SEM) and atomic force microscopy (AFM). The immobilization of anti-p53 antibodies on the electrode surface was investigated by using Fourier-transform infrared spectroscopy (FTIR) and Raman spectroscopy. The change in impedance which formed during the specific interaction between anti-p53 antibody and p53 antigen was used to detect p53. Under optimized experimental conditions, the fabricated immunosensor had a wide linear range of 0.01-2 pg/mL and low detection limit of 3 fg/mL. The fabricated immunosensor had good sensitivity, stability and repeatability. Furthermore, it was successfully applied to analyze p53 in human serum.

Electrochemical ultrasensitive detection of cardiac troponin I using covalent organic frameworks for signal amplification.

The accurate and rapid detection of cardiac troponin I (cTnI) at an early stage can prevent people from getting acute myocardial infarction to a great extent. Here, an ultrasensitive sandwich-type electrochemical immunosensor for the quantitative detection of cTnI is designed based on a novel signal amplification strategy. Gold nanoparticles (Au NPs) doped covalent organic frameworks (COFs) and electron mediator toluidine blue (TB-Au-COFs) behave as labels for achieving signal amplification. Note that polypyrrole modified titanium dioxide nanoparticles (TiO2-PPy) with a high electronic conductivity and a large specific surface area can promote the performance of the immunosensor. For the immobilization of primary antibodies, Au NPs decorated TiO2-PPy (TiO2-PPy-Au) is used as the substrate material to improve electron transfer. The proposed method exhibits a linear range from 0.5 pg mL-1 to 10.0 ng mL-1 and a low detection limit of 0.17 pg mL-1 (S/N = 3). The immunosensor exhibits good stability, acceptable reproducibility and accuracy, indicating potential applications in clinical diagnostics.

A highly selective electrochemical immunosensor based on conductive carbon black and star PGMA polymer composite material for IL-8 biomarker detection in human serum and saliva.

A new approach to enhance the electrochemical performance of biosensor was attempted by using Super P© carbon black/Star polymer composite material. In this study, we developed an electrochemical IL 8 biosensor by modification with a conductive composite including Super P, polyvinylidene fluoride (PVDF) and star polymer (SPGMA) of disposable ITO electrode surface. The Super P carbon black as carbonaceous material had a high conductivity and was used for the enhancement of electron transfer between electrode surface and electrolyte. Anti-IL 8 antibodies were utilized as biorecognition molecules and bound to epoxy groups of star polymer covalently. The chemical characterization of antibody immobilization on this composite was performed by using Fourier-transform infrared spectroscopy (FTIR) and Raman spectroscopy. The characterizations of stepwise modification of this immunosensor were performed by electrochemical techniques such as Electrochemical Impedance Spectroscopy (EIS), Cyclic Voltammetry (CV) and Single Frequency Impedance (SFI); and morphological techniques such as Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). Several variables that affect the immunosensor performance were optimized. Under optimum conditions, a wide linear range 0.01-3 pg/mL and low detection limit 3.3 fg/mL were obtained. Super P-star polymer composite modified immunosensor was easy, sensitive, cheap and reliable analytical method for IL 8 detection. The applicability of the proposed immunosensor to determine IL 8 in saliva and serum samples were examined. The results of biosensor and Enzyme-linked Immunosorbent Assay (ELISA) kit were in compatible. Consequently, it was concluded that the electrochemical immunosensor offers a potential approach for IL 8 detection in clinical applications.

The Display of Single-Domain Antibodies on the Surfaces of Connectosomes Enables Gap Junction-Mediated Drug Delivery to Specific Cell Populations.

Gap junctions, transmembrane protein channels that directly connect the cytoplasm of neighboring cells and enable the exchange of molecules between cells, are a promising new frontier for therapeutic delivery. Specifically, cell-derived lipid vesicles that contain functional gap junction channels, termed Connectosomes, have recently been demonstrated to substantially increase the effectiveness of small molecule chemotherapeutics. However, because gap junctions are present in nearly all tissues, Connectosomes have no intrinsic ability to target specific cell types, which potentially limits their therapeutic effectiveness. To address this challenge, here we display targeting ligands consisting of single-domain antibodies on the surfaces of Connectosomes. We demonstrate that these targeted Connectosomes selectively interact with cells that express a model receptor, promoting the selective delivery of the chemotherapeutic doxorubicin to this target cell population. More generally, our approach has the potential to boost cytoplasmic delivery of diverse therapeutic molecules to specific cell populations while protecting off-target cells, a critical step toward realizing the therapeutic potential of gap junctions.

Dual targeting improves capture of ultrasound microbubbles towards activated platelets but yields no additional benefit for imaging of arterial thrombosis.

Platelets can be found on the surface of inflamed and ruptured atherosclerotic plaques. Thus, targeting of activated platelets may allow for molecular imaging of vulnerable atherosclerotic lesions. We here investigated microbubbles (MB) functionalized with the selectin ligand sialyl Lewisa individually (MBsLea) or dually with sLea and an antibody targeting ligand-induced binding sites of the activated GPIIb/IIIa receptor (MBDual). Assessed by in vitro flow chamber, targeted MB exhibited increased adhesion to platelets as compared to MBControl. While MBsLea rolled slowly on the platelets' surface, MBDual enhanced the percentage of firm adhesion. In vivo, MB were investigated by ultrasound in a model of ferric chloride induced non-occlusive carotid artery thrombosis. MBsLea and MBDual revealed a higher ultrasound mean acoustic intensity than MBControl (p < 0.05), however MBDual demonstrated no additional increase in mean signal intensity as compared to MBsLea. The degree of carotid artery stenosis on histology correlated well with the ultrasound acoustic intensity of targeted MB (p < 0.05). While dual targeting of MB using fast binding carbohydrate polymers and specific antibodies is a promising strategy to support adhesion to activated platelets under arterial shear stress, these advantages seem not readily translatable to in vivo models.

Determination of urinary 15α-hydroxyestrogen levels via immunoaffinity extraction.

15α-Hydroxyestrogens (15α-OHEs) are metabolites of the female hormone estradiol. In this study, to discover physiological markers that can be utilized for monitoring fetal conditions and estrogen-induced cancers, we established a method for quantifying 15α-OHEs in rat urine via immunoaffinity column extraction and HPLC-electrochemical detection, and detected 15α-OHEs in urine obtained male rats treated with estradiol. Notably, the standard curves for quantification obtained using the column were linear over a range of 0.5-50ng 15α-OHEs. The accuracy of the analytical method with cleanup was 97-109% for the three kinds of 15α-OHEs examined, and the intra-assay precision of the measured values had a coefficient of variation of ≤20.6%. Therefore, the theoretical limit of quantification was 0.5ng. However, the actual measured values obtained from the urine of male rats indicated that the detection limits were 0.425, 0.103, and 0.047ng for estetrol, 15α-hydroxyestradiol, and 15α-hydroxyestrone, respectively. Our newly established method for measuring 15α-OHE concentrations in urine could facilitate characterization of the in vivo metabolic profile of 15α-OHEs in mammals under various physiological conditions, which could comprise clinical markers for monitoring human fetal health conditions in mammals.