Development of a Hypersensitive Periodate‐Cleavable Amino Acid that is Methionine‐ and Disulfide‐Compatible and its Application in MHC Exchange Reagents for T Cell Characterisation

Incorporation of cleavable linkers into peptides and proteins is of particular value in the study of biological processes. Here we describe the synthesis of a cleavable linker that is hypersensitive to oxidative cleavage as the result of the periodate reactivity of a vicinal amino alcohol moiety. Two strategies directed towards the synthesis of a building block suitable for solid‐phase peptide synthesis were developed: a chemoenzymatic route, involving L ‐threonine aldolase, and an enantioselective chemical route; these led to α,γ‐diamino‐β‐hydroxybutanoic acids in diastereoisomerically mixed and enantiopure forms, respectively. Incorporation of the 1,2‐amino alcohol linker into the backbone of a peptide generated a conditional peptide that was rapidly cleaved at very low concentrations of sodium periodate. This cleavable peptide ligand was applied in the generation of MHC exchange reagents for the detection of antigen‐specific T cells in peripheral blood cells. The extremely low concentration of periodate required to trigger MHC peptide exchange allowed the co‐oxidation of methionine and disulfide residues to be avoided. Conditional MHC reagents hypersensitive to periodate can now be applied without limitations when UV irradiation is undesired or less practical.     

Synthesis of a novel aromatic–aliphatic hyperbranched polyamide and its application in piezoelectric immunosensors

An aromatic–aliphatic AB₂ monomer, 5‐[3‐(4‐aminophenyl)propionylamino]isophthalic acid, prepared from 5‐aminoisophthalic acid and 3‐(4‐nitrophenyl)propionic acid, and the corresponding hyperbranched polyamide were fabricated. Applications in piezoelectric immunosensors as supports for antibody immobilization were studied. The introduction of the hyperbranched polyamide greatly increased the amount of immobilized antibody on the electrode and consequently increased the sensitivity of the complete piezoelectric immunosensor. The investigation of the properties of the sensors indicated that the hyperbranched polyamide was a suitable support for antibody/antigen immobilization. Copyright © 2007 Society of Chemical Industry     

Human Adipose-Derived Mesenchymal Progenitor Cells Engraft into Rabbit Articular Cartilage

Mesenchymal stem cells (MSCs) are known to have the potential for articular cartilage regeneration, and are suggested for the treatment of osteoarthritis (OA). Here, we investigated whether intra-articular injection of xenogeneic human adipose-derived mesenchymal progenitor cells (haMPCs) promoted articular cartilage repair in rabbit OA model and engrafted into rabbit articular cartilage. The haMPCs were cultured in vitro, and phenotypes and differentiation characteristics of cells were evaluated. OA was induced surgically by anterior cruciate ligament transection (ACLT) and medical meniscectomy of knee joints. At six weeks following surgery, hyaluronic acid (HA) or haMPCs was injected into the knee joints, the contralateral knee served as normal control. All animals were sacrificed at the 16th week post-surgery. Assessments were carried out by macroscopic examination, hematoxylin/eosin (HE) and Safranin-O/Fast green stainings and immunohistochemistry. The data showed that haMPC treatment promoted cartilage repair. Signals of human mitochondrial can be directly detected in haMPC treated cartilage. The haMPCs expressed human leukocyte antigen I (HLA-I) but not HLA-II-DR in vivo. These results suggest that intra-articular injection of haMPCs promotes regeneration of articular cartilage in rabbit OA model, and support the notion that MPCs are transplantable between HLA-incompatible individuals.     

Cloning and Characterization of Surface-Localized α-Enolase of Streptococcus iniae,an Effective Protective Antigen in Mice

Streptococcus iniae is a major fish pathogen that can also cause human bacteremia, cellulitis and meningitis. Screening for and identification of protective antigens plays an important role in developing therapies against S. iniae infections. In this study, we indicated that the α-enolase of S. iniae was not only distributed in the cytoplasm and associated to cell walls, but was also secreted to the bacterial cell surface. The functional identity of the purified recombinant α-enolase protein was verified by its ability to catalyze the conversion of 2-phosphoglycerate (2-PGE) to phosphoenolpyruvate (PEP), and both the recombinant and native proteins interacted with human plasminogen. The rabbit anti-rENO serum blockade assay shows that α-enolase participates in S. iniae adhesion to and invasion of BHK-21 cells. In addition, the recombinant α-enolase can confer effective protection against S. iniae infection in mice, which suggests that α-enolase has potential as a vaccine candidate in mammals. We conclude that S. iniae α-enolase is a moonlighting protein that also associates with the bacterial outer surface and functions as a protective antigen in mice.     

氰戊菊酯免疫分析关键试剂人工抗原的合成与应用效果

以氰戊菊酸、间苯氧基苯甲醛为原料,利用酰氯与伯胺的活泼反应,合成了一种含有3个碳原子长度连接臂的氰戊菊酯半抗原,并通过核磁共振鉴定;用活泼酯法将合成的氰戊菊酯半抗原分别与牛血清白蛋白(BSA)和卵清白蛋白(OVA)偶联制备免疫抗原和检测抗原,紫外-可见连续光谱扫描结果显示,所制备的人工抗原的图谱具有载体蛋白和半抗原的特征峰叠加现象,表明偶联成功;人工抗原的免疫结果显示,以BSA偶联物免疫小鼠后获得抗血清效价分别为160 000、140 000和110 000,间接竞争ELISA(酶联免疫吸附分析)测定抗体IC50(半数抑制浓度)值为35.1 μg/L,与其他供试农药的交叉反应率均＜1%,为氰戊菊酯抗体研制及免疫分析检测技术研究提供了关键试剂.     

Cost-effective disposable thiourea film modified copper electrode for capacitive immunosensor

Cost-effective disposable electrodes were fabricated from copper clad laminate, usually used for printed circuit board (PCB) in electronic industries, by using dry film photoresist. Electro-oxidation (anodisation) was employed to obtain a good formation of thiourea film on the electrode surface. The affinity binding pair of carcinoembryonic antigen (CEA) and anti-carcinoembryonic antigen (anti-CEA) was used as a model system. Anti-CEA was immobilized on thiourea film via covalent coupling. This modified electrode was incorporated with a capacitive system for CEA analysis. This capacitive immunosensor provided a linear range between 0.01 and 10 ng ml⁻¹ with a detection limit of 10 pg ml⁻¹. When applied to analyze CEA in serum samples, the results agreed well with the enzyme linked fluorescent assay (ELFA) technique (P > 0.05). The proposed strategy for the preparation of disposable modified copper electrode is very cost effective and simple. Moreover, it provides good reproducibility. This technique can easily be applied to immobilize other biological sensing elements for biosensors development.     

Inhibitory effects of flavonoid glycosides isolated from the peel of Japanese persimmon (Diospyros kaki Fuyu) on antigen-stimulated degranulation in rat basophilic leukaemia RBL-2H3 cells

We found that two distinct flavonoid glycosides isolated from the peel of Japanese persimmon (Diospyros kaki Fuyu), isoquercitrin (Isq) and hyperin (Hyp), are capable of inhibiting antigen-stimulated degranulation in rat basophilic leukaemia RBL-2H3 cells. In order to elucidate the underlying mechanisms, we examined effects of Isq and Hyp on cellular responses induced by antigen stimulation. Treatment with both Isq and Hyp markedly inhibited antigen-stimulated elevation of intracellular free Ca²⁺ concentration and reactive oxygen species (ROS). Isq and Hyp did not affect NADPH oxidase (NOX) activity, but they possessed DPPH radical-scavenging activity similar to that of epigallocatechin gallate, a potent anti-oxidant, Finally, Isq and Hyp showed little or no effects on Ag-stimulated Syk activation or phosphorylation of signalling molecules. These results indicate that inhibition of antigen-stimulated degranulation by Isq and Hyp is mainly due to suppression of intracellular Ca²⁺ elevation, which is caused by direct scavenging of ROS that are generated by NOX. Our findings suggest that Isq and Hyp, isolated from the peel of persimmon, would be beneficial for alleviating symptoms of type I allergy.     

Sensitive electrochemical immunosensor for the detection of cancer biomarker using quantum dot functionalized graphene sheets as labels

Quantum dot (QD) functionalized graphene sheets (GS) were prepared and used as labels for the preparation of sandwich-type electrochemical immunosensors for the detection of a cancer biomarker (i.e., prostate specific antigen (PSA)). The primary anti-PSA antibody was also immobilized onto the GS. The immunosensor displayed a wide range of linear response (0.005-10 ng/mL), low detection limit (3 pg/mL), and good reproducibility, selectivity and stability. The immunosensor was used to detect PSA in patient serum samples with satisfactory results. Thus, this unique immunosensor may provide many applications in clinical diagnosis.     

Evaluation of Chinese hamster ovary cell stability during repeated batch culture for large-scale antibody production

Pharmaceutical manufacturing plants can be operated continuously for several months. It is therefore important to use cells with long-term stability for the production of active ingredients. We investigated the reliability and long-term stability of an antibody-producing cell line. A recombinant Chinese hamster ovary (CHO) cell line was cultivated in spinner flasks and reactors, including a practical production-scale reactor (1600 L), for 109 days to produce monoclonal antibodies against the HM1.24 antigen. During cultivation, the cells remained stable and there was an increase in the rate of cell proliferation, yielding viable cells at high density. A decrease in cell-specific productivity was associated with this increase in the rate of cell proliferation. The cells were genetically stable and other measures of cellular function remained consistent throughout the cultivation period.     

Towards the detection of human papillomavirus infection by a reagentless electrochemical peptide biosensor

In the present work, we report first results about a technology using a conjugated copolymer poly(5-hydroxy-1,4-naphthoquinone-co-5-hydroxy-2-carboxyethyl-1,4-naphthoquinone) acting both as immobilizing and transducing element for reagentless immunosensor, and its application for the detection of HPV infection. It was shown that the reagentless immunosensor was able to detect the interaction between antigenic peptide L1 from HPV-16 major capsid protein, a dominant epitope involved in viral infection as well as in prophylactic vaccine, and the relevant antibody.