A role for the nucleoporin Nup170p in chromatin structure and gene silencing.

Embedded in the nuclear envelope, nuclear pore complexes (NPCs) not only regulate nuclear transport but also interface with transcriptionally active euchromatin, largely silenced heterochromatin, as well as the boundaries between these regions. It is unclear what functional role NPCs play in establishing or maintaining these distinct chromatin domains. We report that the yeast NPC protein Nup170p interacts with regions of the genome that contain ribosomal protein and subtelomeric genes, where it functions in nucleosome positioning and as a repressor of transcription. We show that the role of Nup170p in subtelomeric gene silencing is linked to its association with the RSC chromatin-remodeling complex and the silencing factor Sir4p, and that the binding of Nup170p and Sir4p to subtelomeric chromatin is cooperative and necessary for the association of telomeres with the nuclear envelope. Our results establish the NPC as an active participant in silencing and the formation of peripheral heterochromatin.

A novel inhalable nanobody targeting IL-4Rα for the treatment of asthma.

BACKGROUND: Inhalable biologics represent a promising approach to improve the efficacy and safety of asthma treatment. Although several mAbs targeting IL-4 receptor α chain (IL-4Rα) have been approved or are undergoing clinical trials, the development of inhalable mAbs targeting IL-4Rα presents significant challenges. OBJECTIVE: Capitalizing on the distinctive advantages of nanobodies (Nbs) in maintaining efficacy during storage and administration, we sought to develop a novel inhalable IL-4Rα Nb for effectively treating asthma. METHODS: Three IL-4Rα immunized Nb libraries were used to generate specific and functional IL-4Rα Nbs. LQ036, a bivalent Nb comprising 2 HuNb103 units, was constructed with a high affinity and specificity for human IL-4Rα. The efficacy, pharmacokinetics, and safety of inhaled LQ036 were evaluated in B-hIL4/hIL4RA humanized mice. RESULTS: LQ036 inhibited secreted embryonic alkaline phosphatase reporter activity, inhibited TF-1 cell proliferation, and suppressed phosphorylated signal transducer and activator of transduction 6 in T cells from patients with asthma. Crystal structure analysis revealed a binding region similar to dupilumab but with higher affinity, leading to better efficacy in blocking the signaling pathway. HuNb103 competed with IL-4 and IL-13 for IL-4Rα binding. Additionally, LQ036 significantly inhibited ovalbumin-specific IgE levels in serum, CCL17 levels in bronchoalveolar lavage fluid, bronchial mucous cell hyperplasia, and airway goblet cell hyperplasia in B-hIL4/hIL4RA humanized mice. Inhaled LQ036 exhibited favorable pharmacokinetics, safety, and tissue distribution, with higher concentrations observed in the lungs and bronchi. CONCLUSIONS: These findings from preclinical studies establish the safety and efficacy of inhaled LQ036, underscoring its potential as a pioneering inhalable biologic therapy for asthma.

Nanobody-Based Microfluidic Immunosensor Chip Using Tetraphenylethylene-Derived Covalent Organic Frameworks as Aggregation-Induced Electrochemiluminescence Emitters for the Detection of Thymic Stromal Lymphopoietin.

In this letter, a sensitive microfluidic immunosensor chip was developed using tetrakis(4-aminophenyl)ethene (TPE)-derived covalent organic frameworks (T-COF) as aggregation-induced electrochemiluminescence (AIECL) emitters and nanobodies as efficient immune recognition units for the detection of thymic stromal lymphopoietin (TSLP), a novel target of asthma. The internal rotation and vibration of TPE molecules were constrained within the framework structure, forcing nonradiative relaxation to convert into pronounced radiative transitions. A camel-derived nanobody exhibited superior specificity, higher residual activity and epitope recognition postcuring compared to monoclonal antibodies. Benefiting from the affinity between silver ions (Ag+) and cytosine (C), a double-stranded DNA (dsDNA) embedded with Ag+ was modified onto the surface of TSLP. A positive correlation was obtained between the TSLP concentration (1.00 pg/mL to 4.00 ng/mL) and ECL intensity, as Ag+ was confirmed to be an excellent accelerator of the generation of free radical species. We propose that utilizing COF to constrain luminescent molecules and trigger the AIECL phenomenon is another promising method for preparing signal tags to detect low-abundance disease-related markers.

High performance production process development and scale-up of an anti-TSLP nanobody.

Nanobodies (Nbs) represent a class of single-domain antibodies with great potential application value across diverse biotechnology fields, including therapy and diagnostics. Thymic Stromal Lymphopoietin (TSLP) is an epithelial cell-derived cytokine, playing a crucial role in the regulation of type 2 immune responses at barrier surfaces such as skin and the respiratory/gastrointestinal tract. In this study, a method for the expression and purification of anti-TSLP nanobody (Nb3341) was established at 7 L scale and subsequently scaled up to 100 L scale. Key parameters, including induction temperature, methanol feed and induction pH were identified as key factors by Plackett-Burman design (PBD) and were optimized in 7 L bioreactor, yielding optimal values of 24 °C, 8.5 mL/L/h and 6.5, respectively. Furthermore, Diamond Mix-A and Diamond MMC were demonstrated to be the optimal capture and polishing resins. The expression and purification process of Nb3341 at 100L scale resulted in 22.97 g/L titer, 98.7% SEC-HPLC purity, 95.7% AEX-HPLC purity, 4 ppm of HCP content and 1 pg/mg of HCD residue. The parameters of the scaling-up process were consistent with the results of the optimized process, further demonstrating the feasibility and stability of this method. This study provides a highly promising and competitive approach for transitioning from laboratory-scale to commercial production-scale of nanobodies.

A humanized trivalent Nectin-4-targeting nanobody drug conjugate displays potent antitumor activity in gastric cancer.

BACKGROUND: Gastric cancer represents a highly lethal malignancy with an elevated mortality rate among cancer patients, coupled with a suboptimal postoperative survival prognosis. Nectin-4, an overexpressed oncological target for various cancers, has been exploited to create antibody-drug conjugates (ADCs) to treat solid tumors. However, there is limited research on Nectin-4 ADCs specifically for gastric cancer, and conventional immunoglobulin G (IgG)-based ADCs frequently encounter binding site barriers. Based on the excellent tumor penetration capabilities inherent in nanobodies (Nbs), we developed Nectin-4-targeting Nb drug conjugates (NDCs) for the treatment of gastric cancer. RESULTS: An immunized phage display library was established and employed for the selection of Nectin-4-specific Nbs using phage display technology. Subsequently, these Nbs were engineered into homodimers to enhance Nb affinity. To prolong in vivo half-life and reduce immunogenicity, we fused an Nb targeting human serum albumin (HSA), resulting in the development of trivalent humanized Nbs. Further, we site-specifically conjugated a monomethyl auristatin E (MMAE) at the C-terminus of the trivalent Nbs, creating Nectin-4 NDC (huNb26/Nb26-Nbh-MMAE) with a drug-to-antibody ratio (DAR) of 1. Nectin-4 NDC demonstrated excellent in vitro cell-binding activities and cytotoxic efficacy against cells with high Nectin-4 expression. Subsequent administration of Nectin-4 NDC to mice bearing NCI-N87 human gastric cancer xenografts demonstrated rapid tissue penetration and high tumor uptake through in vivo imaging. Moreover, Nectin-4 NDC exhibited noteworthy dose-dependent anti-tumor efficacy in in vivo studies. CONCLUSION: We have engineered a Nectin-4 NDC with elevated affinity and effective tumor uptake, further establishing its potential as a therapeutic agent for gastric cancer.

Neutrophil extracellular traps: Potential targets for the treatment of rheumatoid arthritis with traditional Chinese medicine and natural products.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease. Abnormal formation of neutrophil extracellular traps (NETs) at the synovial membrane leads to the release of many inflammatory cytokines, including IL-1ß, IL-6, and TNF-α. Elastase, histone H3, and myeloperoxidase, which are carried by NETs, damage the soft tissues of the joints and aggravate the progression of RA. The balance of NET formation coordinates the pro-inflammatory and anti-inflammatory effects and plays a key role in the development of RA. Therefore, when NETs are used as effector targets, highly targeted drugs with fewer side effects can be developed to treat RA without damaging the host immune system. Currently, an increasing number of studies have shown that traditional Chinese medicines and natural products can regulate the formation of NETs through multiple pathways to counteract RA, which shows great potential for the treatment of RA and has a promising future for clinical application. In this article, we review the latest biological progress in understanding NET formation, the mechanism of NETs in RA, and the potential targets or pathways related to the modulation of NET formation by Chinese medicines and natural products. This review provides a relevant basis for the use of Chinese medicines and natural products as natural adjuvants in the treatment of RA.

TROP2-directed nanobody-drug conjugate elicited potent antitumor effect in pancreatic cancer.

BACKGROUND: Pancreatic cancer is a highly aggressive malignancy with limited treatment options and a poor prognosis. Trophoblast cell surface antigen 2 (TROP2), a cell surface antigen overexpressed in the tumors of more than half of pancreatic cancer patients, has been identified as a potential target for antibody-drug conjugates (ADCs). Almost all reported TROP2-targeted ADCs are of the IgG type and have been poorly studied in pancreatic cancer. Here, we aimed to develop a novel nanobody-drug conjugate (NDC) targeting TROP2 for the treatment of pancreatic cancer. RESULTS: In this study, we developed a novel TROP2-targeted NDC, HuNbTROP2-HSA-MMAE, for the treatment of TROP2-positive pancreatic cancer. HuNbTROP2-HSA-MMAE is characterized by the use of nanobodies against TROP2 and human serum albumin (HSA) and has a drug-antibody ratio of 1. HuNbTROP2-HSA-MMAE exhibited specific binding to TROP2 and was internalized into tumor cells with high endocytosis efficiency within 5 h, followed by intracellular translocation to lysosomes and release of MMAE to induce cell apoptosis in TROP2-positive pancreatic cancer cells through the caspase-3/9 pathway. In a xenograft model of pancreatic cancer, doses of 0.2 mg/kg and 1 mg/kg HuNbTROP2-HSA-MMAE demonstrated significant antitumor effects, and a dose of 5 mg/kg even eradicated the tumor. CONCLUSION: HuNbTROP2-HSA-MMAE has desirable affinity, internalization efficiency and antitumor activity. It holds significant promise as a potential therapeutic option for the treatment of TROP2-positive pancreatic cancer.

Preclinical development of a long-acting trivalent bispecific nanobody targeting IL-5 for the treatment of eosinophilic asthma.

BACKGROUND: Eosinophilic asthma is a common subtype of severe asthma with high morbidity and mortality. The cytokine IL-5 has been shown to be a key driver of the development and progression of disease. Although approved monoclonal antibodies (mAbs) targeting IL-5/IL-5R have shown good safety and efficacy, some patients have inadequate responses and frequent dosing results in medication nonadherence. RESULTS: We constructed a novel trivalent bispecific nanobody (Nb) consisting of 3 VHHs that bind to 2 different epitopes of IL-5 and 1 epitope of albumin derived from immunized phage display libraries. This trivalent IL-5-HSA Nb exhibited similar IL-5/IL-5R blocking activities to mepolizumab (Nucala), an approved targeting IL-5 mAb. Surprisingly, this trivalent Nb was 58 times more active than mepolizumab in inhibiting TF-1-cell proliferation. In primate studies, the trivalent IL-5-HSA Nb showed excellent pharmacokinetic properties, and peripheral blood eosinophil levels remained significantly suppressed for two months after a single dose. In addition, the trivalent IL-5-HSA Nb could be produced on a large scale in a P. pastoris X-33 yeast system with high purity and good thermal stability. CONCLUSIONS: These findings suggest that the trivalent bispecific IL-5-HSA Nb has the potential to be a next-generation therapeutic agent targeting IL-5 for the treatment of severe eosinophilic asthma.

Preclinical evaluation of [99mTc]Tc-labeled anti-EpCAM nanobody for EpCAM receptor expression imaging by immuno-SPECT/CT.

PURPOSE: Overexpression of epithelial cell adhesion molecule (EpCAM) plays essential roles in tumorigenesis and tumor progression in almost all epithelium-derived cancer. Monitoring EpCAM expression in tumors can be used for the diagnosis, staging, and prognosis of cancer patients, as well as guiding the individualized treatment of EpCAM-targeted drugs. In this study, we described the synthesis and evaluation of a site-specifically [99mTc]Tc-labeled EpCAM-targeted nanobody for the SPECT/CT imaging of EpCAM expression. METHODS: We first prepared the [99mTc]Tc-HYNIC-G4K; then, it was site-specifically connected to EpCAM-targeted nanobody NB4. The in vitro characteristics of [99mTc]Tc-NB4 were investigated in HT-29 (EpCAM positive) and HL-60 (EpCAM negative) cells, while the in vivo studies were performed using small-animal SPECT/CT in the subcutaneous tumor models and the lymph node metastasis model to verify the specific targeting capacity as well as the potential applications of [99mTc]Tc-NB4. RESULTS: [99mTc]Tc-NB4 displayed a high EpCAM specificity both in vitro and in vivo. SPECT/CT imaging revealed that [99mTc]Tc-NB4 was cleared rapidly from the blood and normal organs except for the kidneys, and HT-29 tumors were clearly visualized in contrast with HL-60 tumors. The uptake value of [99mTc]Tc-NB4 in HT-29 tumors was increased continuously from 3.77 ± 0.39%ID/g at 0.5 h to 5.53 ± 0.82%ID/g at 12 h after injection. Moreover, the [99mTc]Tc-NB4 SPECT/CT could clearly image tumor-draining lymph nodes. CONCLUSION: [99mTc]Tc-NB4 is a broad-spectrum, specific, and sensitive SPECT radiotracer for the noninvasive imaging of EpCAM expression in the epithelium-derived cancer and revealed a great potential for the clinical translation.

Simultaneous Unlocking Optoelectronic and Interfacial Properties of C60 for Ultrasensitive Immunosensing by Coupling to Metal-Organic Framework.

Due to exceptional electron-accepting ability, light-absorption, and a delocalized conjugated structure, buckminsterfullerene (C60) has attracted fascinating interest in the field of organic solar cells. However, poor delocalization and accumulation of electrons for pristine C60 in physiological aqueous solution and difficulties in conjugation with biomolecules limit its extended photovoltaic applications in bioassay. Herein, we reported the noncovalent coupling of C60 to an electronically complementary porphyrin-derived metal-organic framework (PCN-224) with carboxyl-group terminals. Such assembly not only offered a friendly interface for bioconjugation but also resulted in a long-range ordering C60@PCN-224 donor-acceptor system that demonstrated an unprecedented photocurrent enhancement up to 10 times with respect to each component. As an example, by further cooperating with Nanobodies, the as-prepared C60@PCN-224 was applied to a photoelectrochemical (PEC) immunosensor for S100 calcium-binding protein B with by far the most promising detection activities. This work may open a new venue to unlock the great potential of C60 in PEC biosensing with excellent performances.

A novel bispecific nanobody with PD-L1/TIGIT dual immune checkpoint blockade.

Cancer immunotherapy have changed the paradigm of cancer treatment, but there remains a great need for improvement given that less patients with tumors respond to the treatment of PD-1/PD-L1 blockade. TIGIT (also called T cell immunoreceptor with Ig and ITIM domains), a novel immune checkpoint molecule, has been shown a promising target for drug development of immunotherapy. Here we report generation and characterization of a multivalent bispecific antibody (BsAb) that co-targets PD-L1 and TIGIT. The BsAb consists of tetravalent anti-PD-L1 Fc-fusion nanobody (Nb) and tetravalent anti-TIGIT Nb. The parental anti-PD-L1 Nb showed high specificity and affinity to primate PD-L1, the enhanced T cell activity in vitro and anti-tumor activity in vivo. Similarly, the parental anti-TIGIT Nb showed the high specificity and affinity to primate TIGIT and the enhanced T cell activity. Furthermore, we demonstrated that the BsAb retained high blocking activity towards PD-1/PD-L1 or TIGIT/CD155 interaction. The BsAb synergistically enhanced T cell activities in vitro compared to two parental Nbs. Taken together, we obtained a multivalent BsAb blocking biological function of PD-L1 and TIGIT and it is worthy to further study the anti-tumor activities of this BsAb in vivo.

Evaluation of 64Cu radiolabeled anti-hPD-L1 Nb6 for positron emission tomography imaging in lung cancer tumor mice model.

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 humanlungadenocarcinoma 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.

Preclinical development of a novel CD47 nanobody with less toxicity and enhanced anti-cancer therapeutic potential.

BACKGROUND: CD47, the integrin-related protein, plays an important role in immune resistance and escape of tumor cells. Antibodies blocking the CD47/SIRPα signal pathway can effectively stimulate macrophage-mediated phagocytosis of tumor cells, which becomes a promising approach for tumor immunotherapy. Nanobodies (Nbs) derived from camelid animals are emerging as a new force in antibody therapy. RESULTS: HuNb1-IgG4, an innovative anti-CD47 nanobody, was developed with high affinity and specificity. It effectively enhanced macrophage-mediated phagocytosis of tumor cells in vitro and showed potent anti-ovarian and anti-lymphoma activity in vivo. Importantly, HuNb1-IgG4 did not induce the agglutination of human red blood cells (RBCs) in vitro and exhibited high safety for hematopoietic system in cynomolgus monkey. In addition, HuNb1-IgG4 could be produced on a large scale in CHO-S cells with high activity and good stability. Also, we established anti-CD47/CD20 bispecific antibody (BsAb) consisted of HuNb1 and Rituximab, showing more preference binding to tumor cells and more potent anti-lymphoma activity compared to HuNb1-IgG4. CONCLUSIONS: Both of HuNb1-IgG4 and anti-CD47/CD20 BsAb are potent antagonists of CD47/SIRPα pathway and promising candidates for clinical trials.

Blocking the PD-1-PD-L1 axis by a novel PD-1 specific nanobody expressed in yeast as a potential therapeutic for immunotherapy.

PD-1/PD-L1 pathway blocking with antibodies offers a vital and efficient therapeutic strategy to restore T cell-associated antitumor immunity and treats a variety of cancers in clinic. Nanobodies (Nbs) give several advantages over conventional monoclonal antibodies such as size, solubility, stability and costs. Additionally, P. pastoris is a suitable host for Nb production. Herein, we aim to produce and evaluate anti-PD-1 Nb derived from the P. pastoris. Our findings indicated that we successfully established the Nbs phage-displayed library against PD-1 with qualified library capacity and insert ratio. Anti-PD-1 Nb Nb97 was screened through PE-ELISA and flow cytometry. To extend half-life of Nb97, we contracted pPICZɑA-Nb97-Nb97-HSA recombination vector, which was then transformed into the system of P. pastoris X-33. The yield of purified Nb97-Nb97-Human serum albumin (HSA) fused protein (MY2935) reached to 2.3 g/L after 147 h of fermentation. Meanwhile, the blocking effect of MY2935 is similar to that of MY2626 (humanized Nb97-Fc), and MY2935 showed better performance on stimulating the immune function through PD-1 reporter assay. Hence, P. pastoris X-33 expressing and secreting functional anti-PD-1 Nb-HSA fusion protein might be a system of high yield and low cost.

Construction of Anti-hPD-L1 HCAb Nb6 and in Situ 124I Labeling for Noninvasive Detection of PD-L1 Expression in Human Bone Sarcoma.

Immunotherapy is considered the fourth major treatment mode for cancer following surgery, chemotherapy, and radiotherapy. In recent years, tumor immunotherapy has achieved breakthrough progress; therefore, it is important to screen patients to identify those who will respond to tumor immunotherapy. Here, we report the construction of a novel heavy chain-only antibody (HCAb) and its corresponding 124I-labeled probe. Using phage display technology, we generated a novel anti-hPD-L1-specific HCAb named Nb6 (selected from 95 monoclones) with high affinity for hPD-L1. The positron-emitting 124I-labeled hPD-L1-targeted HCAb probe was prepared for further evaluation, and nonradioactive natural iodine (natI)-labeled anti-hPD-L1 Nb6 was synthesized as a reference compound. 125I-anti-hPD-L1 Nb6 uptake in OS-732 cells in vitro can be blocked by the precursor. The binding affinity of 125I-anti-hPD-L1 Nb6 to OS-732 cell lines was 2.19 nM. For in vivo studies, an osteosarcoma OS-732 tumor-bearing mouse model was successfully constructed. Polymerase chain reaction (PCR) and Western blot analyses were performed to confirm the presence of the hPD-L1 gene and antigen in the tumor tissue of the OS-732 mouse model. Biodistribution showed that uptake of 124I-anti-hPD-L1 Nb6 probes at 24 h was 4.43 ± 0.33% ID/g in OS-732 tumor tissues. Tumor lesions can be clearly delineated on micro-PET (positron emission tomography)/CT (computed tomography) imaging 24 h after injection of 124I-anti-hPD-L1 Nb6, while the blocking group shows substantially decreased uptake on imaging. Pathological staining validated hPD-L1 expression on the surface of the tumor cell membrane; thus, 124I-anti-hPD-L1 Nb6 can be used for in vivo noninvasive PET imaging. When administered in tandem, Nb6 and 124I-anti-hPD-L1 Nb6 may provide a novel strategy to clinically screen patients for hPD-L1 to identify those who would benefit from immunotherapy of malignant tumors such as osteosarcoma.

Remodeling Tumor-Associated Macrophages and Neovascularization Overcomes EGFRT790M -Associated Drug Resistance by PD-L1 Nanobody-Mediated Codelivery.

Precision medicine has made a significant breakthrough in the past decade. The most representative success is the molecular targeting therapy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) in non-small-cell lung cancer (NSCLC) with oncogenic drivers, approved by the US Food and Drug Administration (FDA) as first-line therapeutics for substituting chemotherapy. However, the rapidly developed TKI resistance invariably leads to unsustainable treatment. For example, gefitinib is the first choice for advanced NSCLC with EGFR mutation, but most patients would soon develop secondary EGFRT790M mutation and acquire gefitinib resistance. TKI resistance is a severe emergency issue to be solved in NSCLC, but there are a few investigations of nanomedicine reported to address this pressing problem. To overcome EGFRT790M -associated drug resistance, a novel delivery and therapeutic strategy is developed. A PD-L1 nanobody is identified, and first used as a targeting ligand for liposomal codelivery. It is found that simvastatin/gefitinib combination nanomedicine can remodel the tumor microenvironment (e.g., neovascularization regulation, M2-macrophage repolarization, and innate immunity), and display the effectiveness of reversing the gefitinib resistance and enhancing the EGFRT790M -mutated NSCLC treatment outcomes. The novel simvastatin-based nanomedicine provides a clinically translatable strategy for tackling the major problem in NSCLC treatment and demonstrates the promise of an old drug for new application.

Direct Immunoassay for Facile and Sensitive Detection of Small Molecule Aflatoxin B1 based on Nanobody.

Aflatoxin B1 (AFB1 ), one of the most toxic mycotoxins, is classified as a group I carcinogen and ubiquitous in various foods and agriproducts. Thus, accurate and sensitive determination of AFB1 is of great significance to meet the criteria of food safety. Direct detection of AFB1 is difficult by monoclonal antibody (mAb) with large molecular size (≈150 kD) since the target is too small to produce a detectable signal change. Herein, by combining the electrochemical properties of nanomaterials and the advantages of nanobodies, we developed a direct, highly selective and sensitive electrochemical immunosensor for small molecule detection. The proposed immunosensor had a wide calibration range of 0.01 to 100 ng mL-1 and a low detection limit of 3.3 pg mL-1 (S/N=3). Compared with the immunosensor prepared with mAb which was applied in the typical indirect immunoassay, the immunosensor in this work possessed two orders of magnitudes wider linear range and 10-fold more sensitivity. The as-obtained immunosensor was further successfully applied for sensing AFB1 in real samples. This proposed assay would provide a simple, highly sensitive and selective approach for the direct immunoassay of small molecule AFB1 , and is extendable to the development of direct immunosensing systems for other small molecules detection by coupling nanocarbon and nanobody.

Highly Selective and Sensitive Electrochemical Immunoassay of Cry1C Using Nanobody and π-π Stacked Graphene Oxide/Thionine Assembly.

Cry1C is one of the emerging toxin proteins produced by the Bacillus thuringiensis in the genetically modified crops for pest control in agriculture; thus, it is vital to measure the Cry1C level in crops for the healthy and environmental concerns. Current detections of Cry1C mainly rely on instrumental analysis such as high-performance liquid chromatography, which are time-consuming and are generally cost-prohibitive. Herein, a simple nanobodies (Nbs)-based electrochemical immunosensor has been first proposed for highly selective and sensitive detection of Cry1C. The Nbs pair, i.e., Nb51 and Nb54, which bind to different epitopes on Cry1C, was screened out from an immunized Bactrian camel, with an extra benefit of higher stability compared with conventional antibodies. Further, by using a π-π stacked graphene oxide/thionine assembly that had fast electron transfer kinetics as an electroactive label, the immunoreaction that occurred between the two Nbs and Cry1C can be highly sensitively quantified by square wave voltammetry. The linear detection range was from 0.01 to 100 ng·mL-1, and the low detection limit was 3.2 pg·mL-1. This method was further successfully applied for sensing Cry 1C in spiked samples with recoveries ranging from 100.17% to 106.69% and relative standard deviation less than 4.62%. This proposed assay would provide a simple highly sensitive and selective approach for the Cry1C toxin detection and be applicable to be extended to other toxin proteins sensing in foods.

Specific determination of influenza H7N2 virus based on biotinylated single-domain antibody from a phage-displayed library.

The unpredicted spread of avian influenza virus subtype H7N2 in the world is threatening animals and humans. Specific and effective diagnosis and supervision are required to control the influenza. However, the existing detecting methods are laborious, are time-consuming, and require appropriate laboratory facilities. To tackle this problem, we isolated VHH antibodies against the H7N2 avian influenza virus (AIV) and performed an enzyme-linked immunosorbent assay (ELISA) to detect the H7N2 virus. To obtain VHH antibodies with high affinity and specificity, a camel was immunized. A VHH antibody library was constructed in a phage display vector pMECS with diversity of 2.8 × 10(9). Based on phage display technology and periplasmic extraction ELISA, H7N2-specific VHH antibodies were successfully isolated. According to a pairing test, two VHH antibodies (Nb79 and Nb95) with good thermal stability and specificity can recognize different epitopes of H7N2 virus. The capture antibody (Nb79) was biotinylated in vivo, and the detection antibody (Nb95) was coupled with horseradish peroxidase (HRP). Based on biotin-streptavidin interaction, a novel sandwich immune ELISA was performed to detect H7N2. The immunoassay exhibited a linear range from 5 to 100 ng/ml. Given the above, the newly developed VHH antibody-based double sandwich ELISA (DAS-ELISA) offers an attractive alternative to other diagnostic approaches for the specific detection of H7N2 virus.

Molecular mechanisms of system responses to novel stimuli are predictable from public data.

Systems scale models provide the foundation for an effective iterative cycle between hypothesis generation, experiment and model refinement. Such models also enable predictions facilitating the understanding of biological complexity and the control of biological systems. Here, we demonstrate the reconstruction of a globally predictive gene regulatory model from public data: a model that can drive rational experiment design and reveal new regulatory mechanisms underlying responses to novel environments. Specifically, using ∼ 1500 publically available genome-wide transcriptome data sets from Saccharomyces cerevisiae, we have reconstructed an environment and gene regulatory influence network that accurately predicts regulatory mechanisms and gene expression changes on exposure of cells to completely novel environments. Focusing on transcriptional networks that induce peroxisomes biogenesis, the model-guided experiments allow us to expand a core regulatory network to include novel transcriptional influences and linkage across signaling and transcription. Thus, the approach and model provides a multi-scalar picture of gene dynamics and are powerful resources for exploiting extant data to rationally guide experimentation. The techniques outlined here are generally applicable to any biological system, which is especially important when experimental systems are challenging and samples are difficult and expensive to obtain-a common problem in laboratory animal and human studies.