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- 实验方案
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Her2-Functionalized Gold-Nanoshelled Magnetic Hybrid Nanoparticles: a Theranostic Agent for Dual-Modal Imaging and Photothermal Therapy of Breast Cancer
摘要： Targeted theranostic platform that integrates multi-modal imaging and therapeutic function is emerging as a promising strategy for earlier detection and precise treatment of cancer. Herein, we designed targeted gold-nanoshelled poly (lactic-co-glycolic acid) (PLGA) magnetic hybrid nanoparticles carrying anti-human epidermal growth factor receptor 2 (Her2) antibodies (Her2-GPH NPs) for dual-modal ultrasound (US)/magnetic resonance (MR) imaging and photothermal therapy of breast cancer. The agent was fabricated by coating gold nanoshell around PLGA nanoparticles co-loaded with perfluorooctyl bromide (PFOB) and superparamagnetic iron oxide nanoparticles (SPIOs), followed by conjugating with anti-Her2 antibodies. Cell-targeting studies demonstrated receptor-mediated specific binding of the agent to Her2-positive human breast cancer SKBR3 cells, and its binding rate was significantly higher than that of Her2-negative cells (P < 0.001). In vitro, the agent had capabilities for contrast-enhanced US imaging as well as T2-weighted MR imaging with a relatively high relaxivity (r2 = 441.47 mM?1 s?1). Furthermore, the Her2 functionalization of the agent prominently enhanced the US/MR molecular imaging effect of targeted cells by cell-specific binding. Live/dead cell assay and targeted photothermal cytotoxicity experiments confirmed that Her2-GPH NPs could serve as effective photoabsorbers to specifically induce SKBR3 cell death upon near-infrared laser irradiation. In summary, Her2-GPH NPs were demonstrated to be novel targeted theranostic agents with great potential to facilitate early non-invasive diagnosis and adjuvant therapy of breast cancer.
关键词： Anti-Her2 antibody,Photothermal therapy,Ultrasound imaging,Magnetic resonance imaging,Theranostic agent,Breast cancer
更新于2025-09-16 10:30:52
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111In-labeled anti-cadherin17 antibody D2101 has potential as a noninvasive imaging probe for diagnosing gastric cancer and lymph-node metastasis
摘要： Objective Cadherin-17 (CDH17) is a transmembrane protein that mediates cell–cell adhesion and is frequently expressed in adenocarcinomas, including gastric cancer. CDH17 may be an effective diagnostic marker for the staging of gastric cancer. Here, we developed an 111In-labeled anti-CDH17 monoclonal antibody (Mab) as an imaging tracer and performed biodistribution and single-photon emission computed tomography (SPECT)/computed tomography (CT) imaging studies using mice with CDH17-positive gastric cancer xenografts. CDH17 expression in gastric cancer specimens was also analyzed. Methods The cross-reactivity and affinity of our anti-CDH17 Mab D2101 was evaluated by surface plasmon resonance analysis and cell enzyme-linked immunosorbent assay, respectively. Biodistribution and SPECT/CT studies of 111In-labeled D2101 (111In-D2101) were performed. CDH17 expression in gastric cancer specimens was evaluated by immunohistochemistry. Results Surface plasmon resonance analysis revealed that D2101 specifically recognizes human CDH17, but not murine CDH17. The affinity of D2101 slightly decreased as a result of the radiolabeling procedures. The biodistribution study revealed high uptake of 111In-D2101 in tumors (maximum, 39.2 ± 9.5% ID/g at 96 h postinjection), but low uptake in normal organs, including the stomach. Temporal SPECT/CT imaging with 111In-D2101 visualized tumors with a high degree of tumor-to-nontumor contrast. Immunohistochemical analysis revealed that, compared with HER2, which is a potential marker of N-stage, CDH17 had a higher frequency of positivity in specimens of primary and metastatic gastric cancer. Conclusion Our 111In-anti-CDH17 Mab D2101 depicted CDH17-positive gastric cancer xenografts in vivo and has the potential to be an imaging probe for the diagnosis of primary lesions and lymph-node metastasis in gastric cancer.
关键词： Cadherin-17,Radiolabeled antibody,Lymph-node metastasis,SPECT,Gastric cancer
更新于2025-09-16 10:30:52
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A Model for the Binding of Fluorescently Labeled Anti-Human CD4 Monoclonal Antibodies to CD4 Receptors on Human Lymphocytes
摘要： The CD4 glycoprotein is a component of the T cell receptor complex which plays an important role in the human immune response. This manuscript describes the measurement and modeling of the binding of fluorescently labeled anti-human CD4 monoclonal antibodies (mAb; SK3 clone) to CD4 receptors on the surface of human peripheral blood mononuclear cells (PBMC). CD4 mAb fluorescein isothiocyanate (FITC) and CD4 mAb allophycoerythrin (APC) conjugates were obtained from commercial sources. Four binding conditions were performed, each with the same PBMC sample and different CD4 mAb conjugate. Each binding condition consisted of the PBMC sample incubated for 30 min in labeling solutions containing progressively larger concentrations of the CD4 mAb-label conjugate. After the incubation period, the cells were re-suspended in PBS-based buffer and analyzed using a flow cytometer to measure the mean fluorescence intensity (MFI) of the labeled cell populations. A model was developed to estimate the equilibrium concentration of bound CD4 mAb-label conjugates to CD4 receptors on PBMC. A set of parameters was obtained from the best fit of the model to the measured MFI data and the known number of CD4 receptors on PBMC surface. Divalent and monovalent binding had to be invoked for the APC and FITC CD4 mAb conjugates, respectively. This suggests that the mAb binding depends on the size of the label, which has significant implications for quantitative flow cytometry. The study supports the National Institute of Standards and Technology program to develop quantitative flow cytometry measurements.
关键词： fluorescence spectroscopy,PBMC,FITC,antibody binding,CD4 monoclonal antibody,flow cytometry,APC,cooperative binding
更新于2025-09-12 10:27:22
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Fluorometric lateral flow immunoassay for simultaneous determination of three mycotoxins (aflatoxin B1, zearalenone and deoxynivalenol) using quantum dot microbeads
摘要： A fluorometric lateral flow immunoassay (LFA) is described for the simultaneous determination of the mycotoxins aflatoxin B1 (AFB1), zearalenone (ZEN) and deoxynivalenol (DON). The method is based on the use of CdSe/SiO2 quantum dot microbeads (QBs) with a mean diameter of 106 nm. These have strong red luminescence (with excitation/emission peaks at 365/622 nm) which results in enhanced sensitivity. The QBs binding with monoclonal antibodies (mAbs) as the signal probes can react specifically with AFB1, ZEN and DON, respectively. There is an inverse correlation between the fluorescence signal intensity of test line and the analyte content, which can realize the quantitative analysis of analytes within 15 min. The limits of detection in solution are 10, 80 and 500 pg mL?1 for AFB1, ZEN and DON, respectively. Besides, the average recoveries from spiked feed range from 85.5 to 119.0%, and the relative standard deviations are less than 16.4% for both intra- and inter-day assays. The method was used to analyze naturally contaminated feedstuff, and this resulted in a good agreement with data obtained by LC-MS/MS.
关键词： Rapid detection,LC-MS/MS,Food safety,Multiplexed,Immunochromatographic strip,Quantitative analysis,Cereal samples,Antibody,Feedstuff
更新于2025-09-12 10:27:22
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Modified CdS quantum dots as selective turn-on fluorescent nanosensor for detection and determination of methamphetamine
摘要： Surface modification of cadmium sulfide quantum dots (CdS-QDs) with anti-methamphetamine (anti-METH) antibody produced a new turn-on fluorescent nanosensor, anti-METH-CdS-QDs, for detection of methamphetamine. The anti-METH-CdS-QDs nanosensor was obtained readily via the covalent conjugation of mercaptoacetic acid capped CdS-QDs with anti-METH in the presence of N-ethyl-N′-(3-dimethylaminopropyl carbodiimide) (EDC) and Sulfo-N-hydroxysuccinimide (Sulfo-NHS) as coupling agents. The results of FT-IR and energy dispersive X-ray (EDX) spectroscopies confirmed the functionalization of CdS-QDs with anti-METH species. X-ray diffraction (XRD) and transmission electron microscopy (TEM) analyses revealed the crystal phase and microstructure of CdS-QDs in the anti-METH-CdS-QDs. The obtained anti-METH-CdS-QDs exhibited fluorescence enhancement upon addition of methamphetamine molecules which allowed the highly sensitive and selective determination of methamphetamine with detection limit of 0.006 mg/L. Further studies disclosed that this nanosensor has little interaction with other drugs such as codeine and ibuprofen. Hence, it can be utilized as simple, inexpensive, and selective nanosensor for determination of methamphetamine in low concentrations.
关键词： FRET mechanism,Methamphetamine,Anti-METH antibody,Fluorescent nanosensor,CdS quantum dots
更新于2025-09-12 10:27:22
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Evaluation of 64Cu radiolabeled anti-hPD-L1 Nb6 for positron emission tomography imaging in lung cancer tumor mice model
摘要： Recently, we selected a novel anti-hPD-L1-specific HCAb named Nb6 with high affinity (EC50= 0.65 ng/mL) for potential hPD-L1 targeted non-invasive PET imaging. In this research, Nb6 was conjugated with the bifunctional chelator NCS-Bz-NOTA ((2-[(4-Isothiocyanophenyl) methyl]-1,4,7-triazacy-clononane-1,4,7-triacetic acid)) and further labeled with radio-nuclide 64Cu. 64Cu-NOTA-Nb6 was prepared with over 95% labeling yield, over 99% radiochemical purity and 14~16 GBq/μmol specific activity after PD-10 column purification. It shows good stability in 0.01 M PBS and 5% HSA solutions. 64Cu-NOTA-Nb6 has a high binding affinity to 3.60 nM which was tested by human lung adenocarcinoma A549 cell lines. Tumor lesion can be clearly observed from 20 h to 38 h by Micro-PET equipment after 64Cu-NOTA-Nb6 administration. The study revealed that 64Cu-NOTA-Nb6 has good lesion detection ability, high ratios between tumor and non-tumor signal and can specifically target A549 xenografted tumor model. Taken together of good stability, high binding affinity, and tumor detection ability, 64Cu labeled Nb6 is a promising radio-tracer in diagnosing of hPD-L1 overexpression tumor, supposed to monitor PD-L1overexpression tumor progression and guide targeted therapy with PET molecular imaging.
关键词： Lung cancer,PET/CT imaging,64Cu-NOTA-Nb6,heavy chain-only antibody (HCAb),PD-L1
更新于2025-09-12 10:27:22
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Label-free Identification of Antibody-mediated Rejection in Cardiac Allograft Biopsies Using Infrared Spectroscopic Imaging
摘要： Background. Antibody-mediated rejection (AMR) in cardiac allograft recipients remains less well-understood than acute cellular rejection, is associated with worse outcomes, and portends a greater risk of developing chronic allograft vasculopathy. Diffuse immunohistochemical C4d staining of capillary endothelia in formalin-fixed, paraffin-embedded right ventricular endomyocardial biopsies is diagnostic of immunopathologic AMR but serves more as a late-stage marker. Infrared (IR) spectroscopy may be a useful tool in earlier detection of rejection. We performed mid-IR spectroscopy to identify a unique biochemical signature for AMR. Methods. A total of 30 posttransplant formalin-fixed paraffin-embedded right ventricular tissue biopsies (14 positive for C4d and 16 negative for C4d) and 14 native heart biopsies were sectioned for IR analysis. Infrared images of entire sections were acquired and regions of interest from cardiomyocytes were identified. Extracted spectra were averaged across many pixels within each region of interest. Principal component analysis coupled with linear discriminant analysis and predictive classifiers were applied to the data. Results. Comparison of averaged mid-IR spectra revealed unique features among C4d-positive, C4d-negative, and native heart biopsies. Principal component analysis coupled with linear discriminant analysis and classification models demonstrated that spectral features from the mid-IR fingerprint region of these 3 groups permitted accurate automated classification into each group. Conclusions. In cardiac allograft biopsies with immunopathologic AMR, IR spectroscopy reveals a biochemical signature unique to AMR compared with that of nonrejecting cardiac allografts and native hearts. Future study will focus on the predictive capabilities of this IR signature.
关键词： infrared spectroscopy,Antibody-mediated rejection,C4d,cardiac allograft,biochemical signature
更新于2025-09-12 10:27:22
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Labeling Single Domain Antibody Fragments with Fluorine-18 Using 2,3,5,6-Tetrafluorophenyl 6-[ <sup>18</sup> F]Fluoronicotinate Resulting in High Tumor to Kidney Ratios
摘要： ImmunoPET agents are being investigated to assess the status of epidermal growth factor receptor 2 (HER2) in breast cancer patients with the goal of selecting those likely to benefit from HER2-targeted therapies and monitoring their progress after these treatments. We have been exploring the use of single domain antibody fragments (sdAbs) labeled with 18F using residualizing prosthetic agents for this purpose. In this study, we have labeled two sdAbs that bind to different domains on the HER2 receptor — 2Rs15d and 5F7 — using 2,3,5,6-tetrafluorophenyl 6-[18F]fluoronicotinate ([18F]TFPFN) and evaluated their HER2 targeting properties in vitro and in vivo. The overall decay-corrected radiochemical yield for the synthesis of [18F]TFPFN-2Rs15d and [18F]TFPFN-5F7 was 5.7 ± 3.6% and 4.0 ± 2.0%, respectively. Radiochemical purity of labeled sdAbs was >95%; immunoreactive fractions were about 60% and affinity was in the low nanomolar range. Intracellularly trapped activity from [18F]TFPFN-2Rs15d and [18F]TFPFN-5F7 in HER2-expressing SKOV-3 ovarian and BT474M1 breast carcinoma cells were similar to the sdAbs labeled using the previously validated radioiodination residualizing prosthetic agents N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB) and N-succinimidyl 3-guanidinomethyl-5-[125I]iodobenzoate (iso-[125I]SGMIB). Intracellular activity was about 2-fold higher for radiolabeled 5F7 compared with 2Rs15d for both 18F and 125I. While tumor uptake of both [18F]TFPFN-2Rs15d and [18F]TFPFN-5F7 was comparable to those for the co-administered 125I-labeled sdAb, renal uptake of the 18F-labeled sdAbs was substantially lower. In microPET images, tumor was clearly delineated in SKOV-3 and BT474 xenograft-bearing athymic mice with low levels of background activity in normal tissues except bladder. These results indicate that the [18F]TFPFN prosthetic group could be a valuable reagent for developing sdAb-based immunoPET imaging agents.
关键词： N-Succinimidyl 4-fluorobenzoate (SFB),2,3,5,6-etrafluorophenyl 6-fluoronicotinate (TFPFN),single domain antibody fragment,immunoPET,fluorine-18,HER2
更新于2025-09-11 14:15:04
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Fluorescence immunoassay of E. coli using anti-lipopolysaccharide antibodies isolated from human serum
摘要： In this work, the gram-negative bacterium Escherichia coli strain BL21(DE3) (with lipopolysaccharide (LPS) in its outer membrane) and its modified ClearColi? strain (lacking LPS) were used for the separation of anti-LPS antibodies from human serum by the following steps: (1) binding of the serum proteins to BL21(DE3); (2) dissociation of the bound proteins (including anti-LPS antibodies) from BL21(DE3) with acid; (3) filtering of the dissociated proteins using ClearColi to remove unwanted proteins; and (4) separation of the antibody fraction by protein-A column chromatography. The binding properties of the separated antibodies were analyzed by fluorescence-activated cell sorting to confirm their selective binding to LPS on the outer membrane of BL21(DE3), and by thermophoretic immunoassay to estimate their dissociation constant. The in vitro applicability of the separated anti-LPS antibodies was demonstrated through a fluorescence assay of BL21(DE3), after immobilizing the antibodies onto a modified microplate surface. The electrochemical detection of BL21(DE3) was also achieved after immobilizing the anti-LPS antibodies onto a gold electrode.
关键词： Separation,Anti-LPS antibody,LPS,ClearColi,Human serum
更新于2025-09-10 09:29:36
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Biocompatible Gold Nanorod Conjugates for Preclinical Biomedical Research
摘要： Gold nanorods with a peak absorption wavelength of 760 nm were prepared using a seed-mediated method. A novel protocol has been developed to replace hexadecyltrimethylammonium bromide on the surface of the nanorods with 16-mercaptohexadecanoic acid and metoxy-poly(ethylene glycol)-thiol, and the monoclonal antibody HER2. The physical chemistry properties of the conjugates were monitored through optical and zeta-potential measurements to confirm surface chemistry changes. The efficiency of the modifications was quantified through measurement of the average number of antibodies per gold nanorod. The conjugates were investigated for different cells lines: BT-474, MCF7, MCF10, MDCK, and fibroblast. The results show successful cell accumulation of the gold nanorod HER2 conjugates in cells with HER2 overexpression. Incubation of the complexes in heparinized mouse blood demonstrated the low aggregation of the metallic particles through stability of the spectral properties, as verified by UV/VIS spectrometry. Cytotoxicity analysis with LDH release and MTT assay confirms strong targeting and retention of functional activity of the antibody after their conjugation with gold nanorods. Silver staining confirms efficient specific binding to BT-474 cells even in cases where the nanorod complexes were incubated in heparinized mouse blood. This is confirmed through in vivo studies where, following intravenous injection of gold nanorod complexes, silver staining reveals noticeably higher rates of specific binding in mouse tumors than in healthy liver. The conjugates are reproducible, have strong molecular targeting capabilities, have long term stability in vivo and can be used in pre-clinical applications. The conjugates can also be used for molecular and optoacoustic imaging, quantitative sensing of biological substrates, and photothermal therapy.
关键词： Metallic nanoparticles,Gold nanorod fabrication,Cell particle targeting,Gold surface modification,Biocompatible targeting agent,Antibody conjugation
更新于2025-09-10 09:29:36