Characteristics of monoclonal antibodies against human thyroid peroxidase for use in immunoaffinity chromatography and immunoassays

Two types of monoclonal antibodies (MABs) against human thyroid peroxidase (TPO) have been obtained, which interact with spatially separated conformational epitopes of the antigen (K _a values are in the range 10⁸–10⁹ M^?1). The binding site of MAB F8 is in the immunodominant region of the TPO molecule, in the vicinity of the autoantigenic determinants, whereas the epitope specific for MAB A1 lies outside this location. Both MABs retain the ability to form immune complexes after solid-phase immobilization and chemical modification with a biotin derivative. The above properties suggest that MABs A1 and F8 may be used in immunoaffinity chromatography (isolation and purification of TPO from natural sources) and immunoassays for determinations of TPO (in biological fluids) and TPO autoantibodies (in human blood serum).     

Immunoaffinity chromatography of human thyroid peroxidase: The stability of the three-dimensional structure and immunoreactivity of antigen and antibodies under various elution conditions

Actions of various chemical agents modeling immunoaffinity chromatography elution conditions caused structural changes of the components of human thyroid peroxidase (TPO) complexes with monoclonal antibodies (MABs) F8 and A1 whose antigenic determinants have a conformational nature and are located in the immunodominant region and a peripheral region of TPO, respectively. These changes became apparent in the circular dichroism and fluorescence spectra of TPO and both MABs as well as in the immunoassay. The effectiveness of the chemical reagents with respect to TPO desorption from an immobilized MAB decreased in the following order: 0.2 M ammonia (pH 11.5) > 0.1 M lithium 3,5-diiodosalycilate > 0.1 M glycine-HCl (pH 2.5) > 1 M NaI > 30% propylene glycol + 1 M NaCl > 30% propylene glycol > 1 M NaCl. At pH 11.5, the three-dimensional structure and immunoreactivity of TPO retained completely and only minor alterations of MAB analogical parameters took place, thus providing a high yield of the functional active human TPO and favoring repeated use of the immobilized MABs in immunoaffinity chromatography. The results may be used as a strategy for the optimization of various protein antigens immunoaffinity chromatography.     

C-terminal fragment of human laminin-binding protein contains a receptor domain for Venezuelan equine encephalitis and tick-borne encephalitis viruses

Polyclonal and monoclonal antibodies (MABs) to human laminin-binding protein (LBP) can efficiently block the penetration of some alphaand flaviviruses into the cell. A panel of 13 types of MABs to human recombinant LBP was used for more detailed study of the mechanism of this process. Competitive analysis has shown that MABs to LBP can be divided into six different competition groups. MABs 4F6 and 8E4 classified under competition groups 3 and 4 can inhibit the replication of Venezuelan equine encephalitis virus (VEEV), which is indicative of their interaction with the receptor domain of LBP providing for binding with virions. According to enzyme immunoassay and immunoblotting data, polyclonal anti-idiotypic antibodies to MABs 4F6 and 8E4 modeling paratopes of the LBP receptor domain can specifically interact with VEEV E2 protein and tick-borne encephalitis virus (TBEV) E protein. Mapping of binding sites of MABs 4F6 and 8E4 with LBP by constructing short deletion fragments of the human LBP molecule has shown that MAB 8E4 interacts with the fragment of amino acid residues 187–210, and MAB 4F6 interacts with the fragment of residues 263–278 of LBP protein, which is represented by two TEDWS peptides separated by four amino acid residues. This suggested that the LBP receptor domain interacting with VEEV E2 and TBEV E viral proteins is located at the C-terminal fragment of the LBP molecule. A model of the spatial structure of the LBP receptor domain distally limited by four linear loops (two of which are represented by experimentally mapped regions of amino acid residues 187–210 and 263–278) as well as the central β-folded region turning into the α-helical site including residues 200–216 of the LBP molecule and providing for the interaction with the laminin-1 molecule has been proposed.     

Monoclonal antibodies to the collagen binding domain of human plasma fibronectin

Five independent hybrids producing monoclonal antibodies to human plasma fibronectin have been obtained by fusing P3/X63-Ag8 myeloma cells with immune mouse splenocytes. The specificity of these monoclonal antibodies (MABs) for fibronectin was demonstrated by three independent tests: binding to the purified soluble molecule, immunofluorescence staining of insoluble extracellular matrices produced by endothelial cells in vitro, immunostaining of fibronectin tryptic peptides after separation on SDS-PAGE and transfer to nitrocellulose sheets. Two antibodies (MAB 29 and 52) recognized selectively human fibronectin while the others (MAB 5, 30 and 59) reacted also with plasma fibronectin from calf, hamster and chicken. Four distinct epitopes were recognized by the MABs studied. MAB 5, 30, 52 and 59 reacted with distinct antigenic sites, while MAB 29 and 52 bind to the same site. Antigenic fragments were identified by immunostaining of fibronectin tryptic peptides. MAB 5 reacted with a collagen binding fragment with a molecular weight of 120 K. In addition, each of the MAB 29, 30, 52 and 59 reacted with peptides with a molecular weight of 40 K that bind to gelatin. Since these antibodies do not inhibit fibronectin-collagen interaction, it is concluded that their corresponding epitopes are clustered in a region close, but not coincident, to the collagen binding site of fibronectin.     

Cell surface glycoproteins of hepatocytes and hepatoma cells identified by monoclonal antibodies

Eight hybridoma cell lines secreting monoclonal antibodies (MABs) directed to cell surface components of rat hepatocytes were isolated. The antigens of seven MABs were identified as glycosylated plasma membrane proteins. The presence of these glycoproteins on normal hepatocytes and hepatocellular carcinoma cells was analyzed. A semi-quantitative enzyme-linked immunosorbent assay revealed that only two MABs (Be 8.7, Ne 11.3) recognized proteins which were expressed not only in normal liver but also in chemically induced transplantable Morris hepatomas and hepatoma-derived cell lines. The expression of six antigens was found to be sensitive to transformation. The domain specificity of the MABs was determined by indirect immunofluorescence on sections of liver tissue containing neoplastic nodules. Three MABs (Be 8.4, Ne 11.1, Ne 11.3) specifically bound to the sinusoidal domain and two MABs (Be 9.2, De 13.4) to the bile canalicular domain. These five antigens were transformation-sensitive except for the glycoprotein recognized by the MAB Ne 11.3. Three MABs (Be 8.7, Be 9.1, De 13.2) also showed intracellular immunofluorescence. Two of the antigens (Be 9.1, De 13.2) were not present in hepatomas. The relative molar masses (Mr) of the glycoproteins were determined after protein immunoblotting and immunoprecipitation. Four MABs (Be 8.7, Be 9.1, Be 9.2, De 13.4) recognized antigens with a Mr of 110 000 but did not mutually cross-react. The antigen recognized by MAB De 13.4 was identified as the ectoenzyme dipeptidyl peptidase IV (EC 3.4.14.-).     

抗大肠埃希氏菌F41粘附素特异单克隆抗体的研制及其初步应用

应用杂交瘤技术获得7株能稳定分泌F41特异单克隆抗体的杂交瘤细胞系,分别命名为Ll0、B10、C32、B1、E7、E40和B49。在直接凝集试验、酶联免疫吸附试验和间接荧光试验中,这7株单克隆抗体对所试的31株肠道菌中所有F41阳性菌株都发生反应,与F41阴性菌株则无反应性。抗原竞争ELISA试验结果发现,这些单克隆抗体是针对F41粘附素上相同或十分相近的抗原决定簇。采用肢体金标记技术和免疫电镜证实,这些抗原决定簇在F41粘附素的每条纤毛上多次重复出现。体外肠吸附抑制试验表明,7株F41l特异单克隆抗体对B41M菌株具有很强的抑制能力,对B41菌株则必须用F41和K99单克隆抗体同时作用才具有完全的肠吸附抑制效果。本文用直接酶标单克隆抗体建立的酶联免疫斑点试验具有高度的特异性和灵敏性,可广泛用于现场快速诊断。     

Production and characterization of a panel of monoclonal antibodies to Toxoplasma gondii antigens

A representative collection was obtained containing 68 monoclonal antibodies (MAB) to Toxoplasma gondii antigens, which was characterized by the binding with the below fractions of tochizoites in the immune-enzyme assay (IEA) and immunoblotting (IB): membrane (MEM), somatic (water-soluble, SOM) and excretory-secretory (ES). Most of MABs were produced to MEM antigens (43), 6 MABs reacted with the somatic fraction, and 3 MABs reacted with both fractions. Two MABs to ES antigen were detected in the latter group. An analysis of MABs in concurrent IEA and IB revealed the immune-dominant proteins of the MEM and SOM fractions of antibodies to T. gondii tochizoites (p30 and p27, respectively). The presence of 2 non-overlapping antigenic determinants was shown for p30. Further research would detect MABs that could be used in the diagnosis of toxoplasmosis.     

A novel biphasic extractive membrane bioreactor for minimization of membrane-attached biofilms

Extractive membrane bioreactor (EMB) systems offer a means of biologically treating wastewaters, but, like other membrane processes, are constrained by their tendency to be fouled by membrane-attached biofilms (MABs). This study describes a new approach to eradicate MAB formation and accumulation in EMB systems. To this end, an innovative EMB configuration, the biphasic extractive membrane bioreactor (BEMB), has been developed. In BEMB systems, the two main constituents of the EMB process, membrane and bacteria, are kept separated and interact via a suitable recirculating solvent. Nineteen candidate solvents were tested to assess their suitability for BEMB application. Based on the results of the solvent selection, guidelines are provided to screen solvents for BEMB application. BEMB and EMB runs were carried out to demonstrate the effectiveness of BEMB technology in avoiding MAB accumulation and to compare BEMB and EMB performance. A synthetic wastewater containing monochlorobenzene (MCB) was used as a model system. Abiotic BEMB and EMB runs were carried out and used as comparative references for estimating the effect of MAB accumulation on system performance. MAB thickness in the BEMB systems was controlled at 18 microm during 1 month of operation, whereas, in the EMB systems, MAB thickness reached 1250 microm. Analysis of mass transport in EMB and BEMB systems revealed that the high affinity of the permeating molecules for the solvent may contribute to a reduction in shell-side mass transfer resistance. This reduction of shell-side mass transfer resistance and the absence of MAB accumulation led to overall mass transfer coefficients of about sevenfold greater (4.5 x 10(-5) m s(-1)) in the BEMB system than in the EMB system (0.6 x 10(-5) m s(-1)).     

Identification of novel small molecule enhancers of protein production by cultured mammalian cells

Shortcut nitrogen removal, that is, removal via formation and reduction of nitrite rather than nitrate, has been observed in membrane-aerated biofilms (MABs), but the extent, the controlling factors, and the kinetics of nitrite formation in MABs are poorly understood. We used a special MAB reactor to systematically study the effects of the dissolved oxygen (DO) concentration at the membrane surface, which is the biofilm base, on nitrification rates, extent of shortcut nitrification, and microbial community structure. The focus was on anoxic bulk liquids, which is typical in MAB used for total nitrogen (TN) removal, although aerobic bulk liquids were also studied. Nitrifying MABs were grown on a hollow-fiber membrane exposed to 3 mg N/L ammonium. The MAB intra-membrane air pressure was varied to achieve different DO concentrations at the biofilm base, and the bulk liquid was anoxic or with 2 g m(-3) DO. With 2.2 and 3.5 g m(-3) DO at the biofilm base, and with an anoxic bulk-liquid, the ammonium fluxes were 0.75 and 1.0 g N m(-2) day(-1), respectively, and nitrite was the main oxidized nitrogen product. However, with membrane DO of 5.5 g m(-3), and either zero or 2 g m(-3) DO in the bulk, the ammonium flux was around 1.3 g N m(-2) day(-1), and nitrate flux increased significantly. For all experiments, the cell density of ammonium oxidizing bacteria (AOB) was relatively uniform throughout the biofilm, but the density of nitrite oxidizing bacteria (NOB) decreased with decreasing biofilm DO. Among NOB, Nitrobacter spp. were dominant in biofilm regions with 2 g m(-3) DO or greater, while Nitrospira spp. were dominant in regions with less than 2 g m(-3) DO. A biofilm model, including AOB, Nitrobacter spp., and Nitrospira spp., was developed and calibrated with the experimental results. The model predicted the greatest extent of nitrite formation (95%) and the lowest ammonium oxidation flux (0.91 g N m(-2) day(-1)) when the membrane DO was 2 g m(-3) and the bulk liquid was anoxic. Conversely, the model predicted the lowest extent of nitrite formation (40%) and the highest ammonium oxidation flux (1.5 g N m(-2) day(-1)) when the membrane-DO and bulk-DO were 8 g m(-3) and 2 g m(-3), respectively. The estimated kinetic parameters for Nitrospira spp., revealed a high affinity for nitrite and oxygen. This explains the dominance of Nitrospira spp. over Nitrobacter spp. in regions with low nitrite and oxygen concentrations. Our results suggest that shortcut nitrification can effectively be controlled by manipulating the DO at the membrane surface. A tradeoff is made between increased nitrite accumulation at lower DO, and higher nitrification rates at higher DO.     

Effects of monoclonal antibodies on α-staphylotoxin action against erythrocytes and model phospholipid membranes

It was found that one of twenty tested monoclonal antibodies (MABs) existed which drastically enhanced ability of Staphylococcus aureus α-tosin (ST) to both lysis of human erythrocytes and increase of planar phospholipid bilayer conductance more than 10 and 1000 times respectively. Other 19 MABs possessed only neutralized effect. The activation could only be observed if the activating MAB (AMAB) interacted with ST in solution but not in membrane. The one molecule of AMAB was able to activate approximately 2–4 molecules of ST. It was assumed that this activation was a result of the AMAB-induced transition of ST from a hydrophilic to an amphiphilic form. The activation could not be observed when the activity of AMAB/ST mixtures was tested on highly sensitive rabbit erythrocytes. All the tested MABs (including AMAB) were able to inhibit the ST-induced lysis of rabbit erythrocytes. The activating effects of AMAB on ST action in BLM and in human erythrocytes as well as their inhibiting influence on the ability of toxin to cause a lysis of rabbit erythrocytes indicate the presence of an ST-specific receptor on the membrane of rabbit erythrocytes.     

Detection of time-dependent and oxidatively induced antigens of bovine liver rhodanese with monoclonal antibodies

Rhodanese has been extensively utilized as a model protein for the study of enzyme structure-function relationships. An immunological study of conformational changes occurring in rhodanese as a result of oxidation or thermal inactivation was conducted. Three monoclonal antibodies (MABs) to rhodanese were produced. Each MAB recognized a unique epitope as demonstrated by binding of the MABs to different proteolytic fragments of rhodanese. While none of the MABs significantly bound native, soluble, sulfur-substituted bovine rhodanese, as indicated in indirect enzyme-linked immunosorbent assay experiments, each MAB was immunoadsorbed from solution by soluble rhodanese as a function of the time rhodanese was incubated at 37 degrees C. Thus, as rhodanese was thermally inactivated, conformational changes resulted in the expression of three new epitopes. Catalytic conformers demonstrated different rates of thermally induced antigen expression. Each MAB also recognized epitopes expressed when rhodanese was immobilized on microtiter plates at 37 degrees C. Two conformers resulting from oxidation of rhodanese by hydrogen peroxide were identified immunologically. All MABs recognized rhodanese that was oxidized with peroxide and allowed to undergo a secondary cyanide-dependent reaction which also resulted in a fluorescence shift and increased proteolytic susceptibility. Only one MAB was capable of recognizing an epitope expressed when rhodanese was oxidized with peroxide and then separated from the reactants to prevent induction of the secondary conformational changes.     

Stratification of activity and bacterial community structure in biofilms grown on membranes transferring oxygen

Previous studies have shown that membrane-aerated biofilm (MAB) reactors can simultaneously remove carbonaceous and nitrogenous pollutants from wastewater in a single reactor. Oxygen is provided to MABs through gas-permeable membranes such that the region nearest the membrane is rich in oxygen but low in organic carbon, whereas the outer region of the biofilm is void of oxygen but rich in organic carbon. In this study, MABs were grown under similar conditions but at two different fluid velocities (2 and 14 cm s(-1)) across the biofilm. MABs were analyzed for changes in biomass density, respiratory activity, and bacterial community structure as functions of biofilm depth. Biomass density was generally highest near the membrane and declined with distance from the membrane. Respiratory activity exhibited a hump-shaped profile, with the highest activity occurring in the middle of the biofilm. Community analysis by PCR cloning and PCR-denaturing gradient gel electrophoresis of 16S rRNA genes demonstrated substantial stratification of the community structure across the biofilm. Population profiles were also generated by competitive quantitative PCR of gene fragments specific for ammonia-oxidizing bacteria (AOB) (amoA) and denitrifying bacteria (nirK and nirS). At a flow velocity of 14 cm s(-1), AOB were found only near the membrane, whereas denitrifying bacteria proliferated in the anoxic outer regions of the biofilm. In contrast, at a flow velocity of 2 cm s(-1), AOB were either not detected or detected at a concentration near the detection limit. This study suggests that, under the appropriate conditions, both AOB and denitrifying bacteria can coexist within an MAB.     

Distribution and composition of extracellular polymeric substances in membrane-aerated biofilm

Extracellular polymeric substances (EPS) are one of the main components of the biofilm and perform important functions in the biofilm system. In this study, two membrane-aerated biofilms (MABs) were developed for the thin and thick biofilms under different surface loading rates (SLRs). Supplies of oxygen and substrates in the MAB were from two opposite directions. This counter diffusion of nutrients resulted in a different growth environment, in contrast to conventional biofilms receiving both oxygen and substrates from the same side. The compositions, distributions and physicochemical properties (solubility and bindability) of EPS in the MABs of different thicknesses under different SLRs were studied. The effect of dissolved oxygen (DO) concentration within the MAB on EPS properties and distribution was investigated. Experimental results showed the different biofilm thicknesses produced substantially different profiles of EPS composition and distribution. Soluble proteins were more dominant than soluble polysaccharides in the inner aerobic layer of the biofilms; in contrast, bound proteins were greater than bound polysaccharides in the outer anoxic or anaerobic layer of the biofilms. The biofilm-EPS matrix consisted mainly of bound EPS. Bound EPS exhibited a hump-shaped profile with the highest content occurring in an intermediate region in the thin MAB and relatively more uniformly in the one half of the biofilm close to the membrane side and then declined towards the biofilm-liquid interface in the thick MAB. The profiles of soluble EPS presented a similar declining trend from the membrane towards the outer region in both thin and thick MABs. The study suggested that not only EPS composition but also EPS distribution and properties (solubility and bindability) played a crucial role in controlling the cohesiveness and maintaining the structural stability and stratification of the MABs.     

Control of membrane-attached biofilms using surfactants

The effect of surfactants on membrane-attached biofilms (MABs) was studied in a lab-scale extractive membrane bioreactor (EMB). Twenty-two surfactants were screened for their potential of increasing the cell wall negative charge (i.e. the electrostatic repulsion between bacteria) of Burkholderia sp. JS150 bacterial strain. Surfactants resulting in increased bacterial negative charge were further investigated for their effects on MAB population morphology and MAB attachment behaviour. Microscopic investigation of the bacterial population in MABs showed that surfactants affect the development of flagella, suggesting changes in the attachment capability of the JS150 strain in the presence of different surfactants. Among the screened surfactants, teepol showed the best characteristics in relation to the reduction of MAB accumulation, and it was tested in an EMB system for the extraction of monochlorobenzene from a synthetic wastewater. Comparison with a control EMB, operated without surfactants under the same conditions, proved that teepol effectively reduces MAB accumulation on the membrane walls. As a result, the overall mass transfer coefficient in the presence of teepol was 53% higher than in the control EMB.     

Notch signaling regulates myogenic regenerative capacity of murine and human mesoangioblasts

Somatic stem cells hold attractive potential for the treatment of muscular dystrophies (MDs). Mesoangioblasts (MABs) constitute a myogenic subset of muscle pericytes and have been shown to efficiently regenerate dystrophic muscles in mice and dogs. In addition, HLA-matched MABs are currently being tested in a phase 1 clinical study on Duchenne MD patients (EudraCT #2011-000176-33). Many reports indicate that the Notch pathway regulates muscle regeneration and satellite cell commitment. However, little is known about Notch-mediated effects on other resident myogenic cells. To possibly potentiate MAB-driven regeneration in vivo, we asked whether Notch signaling played a pivotal role in regulating MAB myogenic capacity. Through different approaches of loss- and gain-of-function in murine and human MABs, we determined that the interplay between Delta-like ligand 1 (Dll1)-activated Notch1 and Mef2C supports MAB commitment in vitro and ameliorates engraftment and functional outcome after intra-arterial delivery in dystrophic mice. Furthermore, using a transgenic mouse model of conditional Dll1 deletion, we demonstrated that Dll1 ablation, either on the injected cells, or on the receiving muscle fibers, impairs MAB regenerative potential. Our data corroborate the perspective of advanced combinations of cell therapy and signaling tuning to enhance therapeutic efficaciousness of somatic stem cells.Notch signaling consists of a conserved pathway, triggered by physical interaction between one ligand and one receptor, both transmembrane proteins exposed by contacting cells.¹ Notch signaling has been involved in different stages of muscle formation² and regeneration.^3,4 The canonical signaling encompasses five ligands (Dll1/3/4, Jagged1/2) and four receptors (Notch1–4); however, the axis Dll1-Notch1 appears consistently involved during myogenic fate specification, for example, neural crest-driven somite maturation.⁵ Moreover, murine embryos expressing a hypomorphic allele of the Notch ligand Dll1 displayed marked impairment of skeletal muscle formation.⁶ Interestingly, the Notch pathway may exert different effects according to the cell context. Culture on DLL1-coated plastic improved ex vivo proliferation and in vivo engraftment of canine satellite cells.⁷ Expression of the active Notch1 intracellular domain (NICD) robustly committed murine and rat mesenchymal stem cells toward the myogenic fate both in vitro and in vivo.⁸ However, Notch-mediated effects on the regenerative potential of non-satellite resident myogenic cells are still unknown.Mesoangioblasts (MABs) are non-satellite resident myogenic stem cells, able to circulate and regenerate dystrophic skeletal muscles.^9,10 HLA-matched MABs are currently under phase 1 clinical study on Duchenne muscular dystrophy patients (EudraCT #2011-000176-33). In this view, understanding the cell-specific effects and mechanisms of myogenic cues will help improving clinical translation of MAB-based therapies in vivo. Recently, it has been shown that Notch synergizes with Pdgf-bb to convert fetal myoblasts into myogenic pericytes.¹¹ However, knowledge about Notch-triggered effects on the regenerative potency of somatic MABs is still scant, particularly in the contexts of cell–cell (in vitro) and fiber–cell (in vivo) contact.Therefore, we asked whether the Dll1-Notch1 axis regulates the myogenic potential of murine and human MABs and how to tune this pathway to ameliorate in vivo MAB-driven regeneration.     

Effect of bulk liquid BOD concentration on activity and microbial community structure of a nitrifying,membrane-aerated biofilm

Membrane-aerated biofilms (MABs) are an effective means to achieve nitrification and denitrification of wastewater. In this research, microsensors, fluorescence in situ hybridization (FISH), and modeling were used to assess the impact of bulk liquid biological oxygen demand (BOD) concentrations on the activity and microbial community structure of nitrifying MABs. With 1 g m⁻³ BOD in the bulk liquid, the nitrification rate was 1.3 g N m⁻² day⁻¹, slightly lower than the 1.5 g N m⁻² day⁻¹ reported for no bulk liquid BOD. With bulk liquid BOD concentrations of 3 and 10 g m⁻³, the rates decreased to 1 and 0.4 g N m⁻² day⁻¹, respectively. The percent denitrification increased from 20% to 100% when the BOD increased from 1 to 10 g m⁻³ BOD. FISH results indicated increasing abundance of heterotrophs with increasing bulk liquid BOD, consistent with the increased denitrification rates. Modeling was used to assess the effect of BOD on nitrification rates and to compare an MAB to a conventional biofilm. The model-predicted nitrification rates were consistent with the experimental results. Also, nitrification in the MAB was much less sensitive to BOD inhibition than the conventional biofilm. The MAB achieved concurrent nitrification and denitrification, whereas little denitrification occurred in the conventional biofilm.     

Production of a monoclonal antibody specific for a Flavobacterium species isolated from soil

Abstract Hybridomas secreting monoclonal antibodies (MABs) specific for a soil Flavobacterium species (P25) were isolated. The MAB (D10) was used to target P25 using an enzyme-linked immunosorbant assay (ELISA) and indirect immunofluorescence. Cross-reactivity of the MAB with other Gram-negative bacteria (including Flavobacterium spp.) and a number of Gram-positive bacteria was investigated but none were found. Cross-reactivity with other orange/yellow pigmented Gram-negative rods ( Pseudomonas/Flavobacterium type) isolated from the soil into which P25 has been introduced in field experiments was also assessed using a modified colony blotting procedure. None of the indigenous species tested were recognised by the monoclonal antibody, thereby allowing unambiguous identification of P25 in soil. The MAB D10 was shown to recognise P25 growth under low-nutrient or stored under starvation conditions, suggesting that the antigen is a constitutive component of the cell and that the microorganism should be detected in oligotrophic environments such as soil. The pattern of fluorescence of P25 gave a clear indication of the localisation of the antigen in the outer membrane/cell wall region, and this was confirmed by immunogold labelling. Preliminary studies on the limits of detection of P25 using immunofluorescence suggest that densities as low as 20 bacteria g⁻¹ soil can be enumerated.     

TPO模拟肽与人IgG1 Fc片段的融合表达及其生物学特性研究

依据本室获得的人TPO模拟肽序列,合成了该模拟肽的DNA序列,分别连接至4种不同长度的人IgG1 Fc基因片段的5′端,并克隆至质粒表达载体pET28a( ),转化大肠杆菌BL21（DE3）,筛选获得了4种重组工程菌,其中3种分别高效表达了3种不同长度的融合蛋白,而第4种工程菌未表达,表达的3种融合蛋白的分子量分别约为28kD,12kD和12kD。表达量约占菌体蛋白总量的30％左右,纯化获得了3种TPO模拟肽融合蛋白,3种融合蛋白均有较好的体外活性,维持TPO依赖细胞Ba/F3-mp1生长的EC50分别为：13,10,10nmol/L,用血小板减少症小鼠动物模型,测定了它们的体内活性,3种融合蛋白均有升高血小板和缩短血小板恢复时间的功能,分别比TPO模拟肽活性提高了18,8,8倍,而对白细胞及红细胞无显著影响,分别用3种融合蛋白免疫BALB／c小鼠,均未刺激小鼠产生抗TPO模拟肽抗体,并显示了较好的应用潜力。     

Use of monoclonal antibodies for identification of growth-controlling neuropeptides in the mussel Mytilus edulis (Mollusca: Bivalvia)

Monoclonal antibodies were developed against cerebral ganglia (CG) of the mussel Mytilus edulis by the immunization of mice with unpurified homogenates of these organs. The screening protocol of hybridoma was based upon immunohistological observations of cytocentrifugated ganglia cells. A panel of 29 monoclonal antibodies (MABs) specific of CG epitopes was harvested and subsequently used for the immunocytochemical study of CG cells. Several subpopulations of ganglia cells were specifically revealed by MABs. Identification of epitopes involved in growth control was approached via the application of a bioassay allowing the assessment of protein synthesis stimulation. MAB 42 and 46 affected amino acid incorporation induced by CG extract. These results lead to the conclusion that the epitopes recognised by these antibodies are involved in growth control. An immunoenzymatic assay was performed with CG extracts for quantitative analyses of epitopes.     

Imaging Striatal Microglial Activation in Patients with Parkinson’s Disease

This study investigated whether the second-generation translocator protein 18kDa (TSPO) radioligand, [¹⁸F]-FEPPA, could be used in neurodegenerative parkinsonian disorders as a biomarker for detecting neuroinflammation in the striatum. Neuroinflammation has been implicated as a potential mechanism for the progression of Parkinson’s disease (PD). Positron Emission Tomography (PET) radioligand targeting for TSPO allows for the quantification of neuroinflammation in vivo. Based on genotype of the rs6791 polymorphism in the TSPO gene, 16 mixed-affinity binders (MABs) (8 PD and age-matched 8 healthy controls (HCs)), 16 high-affinity binders (HABs) (8 PD and age-matched 8 HCs) and 4 low-affinity binders (LABs) (3 PD and 1 HCs) were identified. Total distribution volume (V_T) values in the striatum were derived from a two-tissue compartment model with arterial plasma as an input function. There was a significant main effect of genotype on [¹⁸F]-FEPPA V_T values in the caudate nucleus (p = 0.001) and putamen (p < 0.001), but no main effect of disease or disease x genotype interaction in either ROI. In the HAB group, the percentage difference between PD and HC was 16% in both caudate nucleus and putamen; in the MAB group, it was -8% and 3%, respectively. While this PET study showed no evidence of increased striatal TSPO expression in PD patients, the current findings provide some insights on the possible interactions between rs6791 polymorphism and neuroinflammation in PD.