Establishment of Monoclonal Antibodies Specific for Bacillus subtilis DB9011

Bacillus subtilis DB9011 is a strain with useful functions for agriculture. To establish a method for the discrimination of this strain from others, monoclonal antibodies (MAbs) were prepared. Although two established MAbs (MAb9B6 and MAb14D2) cross-react with some other Bacillus strains in ELISA, only B. subtilis DB9011 vegetative cells are recognized by both MAbs. MAb14D2 recognizes flagellin, a 34-kDa unit protein of flagella. The two MAbs established will provide powerful tools with which detailed analysis of this bacterial strain can be obtained under environmental conditions.     

Flagellin as a Biomarker for Bacillus subtilis Strains; Application to the DB9011 Strain and the Study of Interspecific Diversity in Amino-acid Sequences

Polymerase chain reaction (PCR) for the flagellin central domain coding region (FCD-PCR) was applied to the detection and discrimination of Bacillus subtilis DB9011, a strain with useful functions in agriculture. Cross-reactions were observed in 4 B. subtilis strains with similar flagellin genes (hag). Alignment of partial amino-acid sequences of flagellin and the results of PCR for the 16S/23S rRNA spacer in 11 B. subtilis strains suggested the presence of a group including strains with antifungal activity (DB9011 and others).     

Flagellin as a biomarker for Bacillus subtilis strains; application to the DB9011 strain and the study of interspecific diversity in amino-acid sequences.

Polymerase chain reaction (PCR) for the flagellin central domain coding region (FCD-PCR) was applied to the detection and discrimination of Bacillus subtilis DB9011, a strain with useful functions in agriculture. Cross-reactions were observed in 4 B. subtilis strains with similar flagellin genes (hag). Alignment of partial amino-acid sequences of flagellin and the results of PCR for the 16S/23S rRNA spacer in 11 B. subtilis strains suggested the presence of a group including strains with antifungal activity (DB9011 and others).     

Calcium requirements of residual protease in Bacillus subtilis DB104

Summary Bacillus subtilis DB104, a double mutant which does not synthesize neutral or alkaline proteases, was shown to exhibit some residual proteolytic activity when grown in both batch and continuous cultures. A major protein component responsible for about 70% of extracellular residual protease activity was reversibly deactivated by removal of calcium.     

Mouse monoclonal antibodies specific for endothelial cells

Summary Balb/c mice were immunized with a human endothelial cell pool. Spleen cells were then fused with a NS-0 hybridoma cell line. A number of hybridomas secreted antibodies that reacted with the immunizing endothelial cell pool as well as with every other tested umbilical cord vein~derived human endothelial cell. These monoclonal antibodies also stained pig, rabbit and ox aortic endothelial cells indicating their specificity for this cell type. Five of 16 monoclonal antibodies additionally reacted with human fibroblasts (HFIB). The produced monoclonal antibodies did not recognize FVIIIRAG or MHC determinants. They can therefore be regarded as additional and reliable markers for endothelial cells in vitro.     

产青霉素酶枯草芽孢杆菌发酵条件的优化

目的:对一株产青霉素酶的重组枯草芽孢杆菌(Bacillus subtilis DB104/pWB-pemp)的摇瓶发酵条件进行优化.方法:利用单因素实验及正交实验等方法考查重组菌摇瓶发酵条件,研究不同浓度的碳源、氮源、金属离子、磷酸盐及不同pH、接种量、装液量和温度等条件对产青霉素酶的影响.结果:重组菌株摇瓶最适发酵条件为pH 7.0、接种量3％、装液量14％、发酵温度37℃,培养基成分为2％蔗糖、3.5％酵母膏、0.1mmol/L镁离子、0.4％磷酸盐.结论:最优条件下,发酵36h,产酶活力达到2227.8U/mL,为先前研究结果(1 580 U/mL)的1.41倍,为重组菌的大规模发酵生产奠定了基础.     

Production of monoclonal antibodies specific for Eimeria tenella microgametocytes

A panel of 4 monoclonal antibodies specific for Eimeria tenella, the causative agent of cecal coccidiosis of birds in the genus Gallus, was produced by standard techniques. The indirect fluorescent antibody (IFA) test demonstrated specificity of these 4 antibodies for the microgametocytes. Hybridoma TIA3B9 secreted a monoclonal antibody of subisotype IgG2b that was used throughout the course of this study. Immunologic potency of this antibody was demonstrated by in vitro experiments that revealed a greater than 50% reduction in oocyst production, indicating an apparent inhibition of fertilization.     

An endonuclease activity in Bacillus subtilis specific for UV-irradiated DNA

An enzyme activity specific for UV-DNA¹ was found in the extract of $\text{Bacillus subtilis}$(Marburg 168). The enzyme preparation obtained from the extract by ammonium sulfate precipitation acts on UV-DNA endonucleolytically and induces single strand breaks. The number of single strand breaks introduced in DNA is proportional to UV dose.     

An intracellular endonuclease of Bacillus subtilis specific for single-stranded DNA.

We have fractionated from extracts of Bacillus subtilis the DNase activity specific for single-stranded DNA; the activity separates in two main fractions on Sephadex G-200, a larger one (Mr greater than 400 000) and a smaller one (Mr approximately 30 000). We have purified the smaller, more abundant fraction nearly 3000-fold. The purified enzyme has a pH optimum close to 8, is activated by Ca2+, and is inhibited by EDTA; the enzyme hydrolyses single-stranded DNA at a rate approximately 40 times greater than double-stranded DNA. The mode of action is endonucleolytic on both substrates, but the possiblility that the two activities may reside on different molecules is not ruled out. The products have 5'-P and 3'-OH ends. The enzyme is different from those purified from the culture media of the same organism in several respects; the latter are all extracellular enzymes, they are not specific for single-stranded DNA (except one) and have all an exonucleolytic mode of action.     

Characterization of high affinity monoclonal antibodies specific for chlamydial lipopolysaccharide

Pathogens belonging to the genus Chlamydia contain lipopolysaccharide with a 3-deoxy-D- manno- oct-2-ulosonic acid (Kdo) trisaccharide of the sequence alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo. This lipopolysaccharide is recognized in a genus-specific pattern by murine monoclonal antibodies (mAbs), S25-23 and S25-2 (both IgG1kappa), which bind as the minimal structures the trisaccharide and the terminal Kdo-disaccharide, respectively. The variable domains of these mAbs were reverse transcribed from mRNA which was isolated from hybridomas and cloned as single-chain variable fragments (scFvs) in E.coli TG1. The kinetics of binding of whole antibodies, Fab fragments and scFvs to natural and synthetically modified ligands were determined by surface plasmon resonance (SPR) using synthetic neoglycoconjugates. As examples of an antibody-carbohydrate interaction involving anionic carboxyl groups on the ligand, we report that the affinities of these antibodies are higher than usually observed in carbo-hydrate-protein interactions (K(D)of 10(-3)to 10(-5)M). SPR analy-ses of monovalent Fab and scFv binding to the natural trisaccharide epitope gave dissociation constants of 770 nM for S25-2 and 350 nM for S25-23, as determined by global fitting (simultaneous fitting of several measurements at different antibody concentrations) of sensorgram data to a one-to-one interaction model. Local fitting (separate fitting of individual sensorgram data at different antibody concentrations) and Scatchard analysis of the data gave kinetic and affinity constants that were in good agreement with those obtained by global fitting. The SPR data also showed that while S25-2 bound well to several Kdo disaccharides and carboxyl-reduced Kdo ligands, S25-23 did not. Identification of amino acids in the complementarity determining regions revealed the presence of a large number of positively charged amino acids which were located towards the center of the combining site, thus suggesting a different recognition mechanism than that observed for neutral ligands. The latter mainly involves aromatic amino acids for hydrophobic stacking inter-actions and hydrogen bonds.     

Production of monoclonal antibodies specific for ganglioside GD3.

Four kinds of anti-GD3 monoclonal antibodies, DSG-1, -2, -3, and -4, of the IgM class were obtained by the immunization of BALB/c mice with enzootic bovine leukosis tumor tissue-derived ganglioside GD3 inserted into liposomes with Salmonella minnesota R595 lipopolysaccharides. The specificities of the monoclonal antibodies obtained were defined by complement-dependent liposome immune lysis assay and by enzyme immunostaining on thin-layer chromatography. The reactivities of the monoclonal antibodies obtained to four ganglioside GD3 variants [GD3(NeuAc-NeuAc), GD3(NeuAc-NeuGc), GD3(NeuGc-NeuAc), and GD3(NeuGc-NeuGc)] were tested. All of the monoclonal antibodies were found to react with GD3(NeuAc-NeuAc) and GD3(NeuAc-NeuGc) but not with GD3(NeuGc-NeuAc) or GD3(NeuGc-NeuGc). Furthermore, various purified glycosphingolipids were used to determine the specificity of these monoclonal antibodies. All 4 antibodies reacted only with ganglioside GD3 [GD3(NeuAc-NeuAc) and GD3(NeuAc-NeuGc)], but not with several gangliosides linking the GalNAc, Gal beta 1-3GalNAc, NeuAc alpha 2-3Gal beta 1-3GalNAc, or NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-3GalNAc residue to the Gal moiety of ganglioside GD3 (GD2, GD1b, GT1b, or GQ1b, respectively), ganglioside GT1a having the same terminal NeuAc alpha 2-8NeuAc alpha 2-3Gal residue as ganglioside GD3, other gangliosides, and neutral glycosphingolipids. These findings suggest that the 4 monoclonal antibodies obtained may be specific for the epitope of NeuAc-alpha 2-8Sia alpha 2-3Gal beta 1-4Glc residue of ganglioside GD3.     

Production and characterization of monoclonal antibodies specific for Mycobacterium bovis

A series of monoclonal antibodies (MAbs), specific for Mycobacterium bovis and BCG strains, were tested extensively for cross-reactivity to a wide range of mycobacterial species using ELISA, Western blotting and dot-blot analysis. The MAbs bound specifically to M. bovis and BCG and showed limited cross-reactivity with some strains of M. tuberculosis. All these MAbs recognized a 22 kDa protein previously termed MPB70, and by competitive ELISA analysis appeared to detect at least three M. bovis-specific determinants on the MPB70 molecule.     

用合成多肽免疫制备尿激酶原单克隆抗体

根据尿激酶原与尿激酶一级结构的区别并结合计算机分子模拟,设计合成了包括尿激酶原Thr152-Glu163肽段的13肽,然后与载体蛋白KLH偶联作为免疫原,用BI林巴细胞融合技术获得了3种尿激酶原特异性单克隆抗体,这3种抗体仅与尿激酶原和合成多肽反应 ,而不与尿激酶及其结构类似物组织型纤溶酶原激活剂,凝血酶,纤维蛋白原反应,琼脂双向免疫扩散实验及酶活性抑制实验表明,3种抗体均为IgG类的IgG1亚类,所有3种抗体均不抑制酶活力,探讨了这组抗体用于尿激酶原结构与功能及其定量,定性分析研究方面的可能性。     

Highly specific labeling of the Bacillus subtilis chromosome terminus

By making use of the sporulation process, the terminus region of the Bacillus subtilis chromosome has been labeled with [3H]thymine in a highly specific manner. The result achieved supports the view that B. subtilis spores contain only completed chromosomes.     

Characterization of anti-Citrobacter 036 specific polysaccharide monoclonal antibodies

Monoclonal mouse antibodies specific for the 0 antigen of Citrobacter 036, a homopolymer of beta (1----2)-linked 4-deoxy-D-arabinohexose, were generated by the hybridoma technique. Balb/c mice were immunized with killed whole-cell vaccine and initial selection of active clones was based on enzyme-linked immunosorbent assay (ELISA) employing purified lipopolysaccharide (LPS). Concentrated culture supernatants from selected hybrid cultures were used to identify 10 0-antigen specific monoclonal antibodies using the multiple criteria of immunoprecipitation of 0 chains and LPS, inhibition by acid hydrolyzed 0 chains in the screening ELISA, and antibody class analysis. Four monoclonal antibodies were chosen for further study using dose-dependent 0-chain inhibition of ELISA and passive hemagglutination, passive hemolysis, and bacterial agglutination titres. When screened with Citrobacter serotypes known to contain the sugar 4-deoxy-D-arabinose, passive hemagglutination tests showed that the two monoclonal antibodies examined possessed titres which could be correlated with the reported 4-deoxy-D-arabinohexose content of the respective LPS's. This sugar is an antigenically important unit of several Citrobacter serotypes as defined by these well-characterized monoclonal antibodies.     

CK-MB特异性单抗的制备方法及其应用

本研究拟建立肌酸激酶同工酶MB(CK-MB)特异性单克隆抗体(m Ab)的研制方法,对抗CK-MB单抗进行评价分类及性质鉴定,并初步建立CK-MB定量检测试剂。以CK-MB抗原免疫BALB/c小鼠,利用常规单抗制备技术,使用间接和捕获ELISA差异筛选法筛选单抗。利用肌酸激酶同工酶(CK-MM/BB/MB)抗原对所制备单抗的抗原识别表位进行鉴定,另通过免疫印迹法及合成CK-MM、CK-BB差异性的线性表位肽鉴定对所制备的单抗进行评价分类。使用双抗体夹心ELISA方法筛选检测CK-MB抗原的配对m Ab,并初步建立CK-MB定量检测试剂。使用74例临床标本初步评价该试剂与罗氏试剂的检测一致性。最终,我们成功筛选到22株稳定分泌抗CK-MB抗体的杂交瘤细胞株,这些单抗可以分为线性、偏构象的CK-MB和CK-MM或者CK-BB交叉的单抗以及与CK-MB特异反应的偏构象型单抗,并使用偏构象型单抗研制出CK-MB定量检测试剂,该试剂与罗氏试剂相关系数r达到0.930 9。综上所述,本研究建立了研制CK-MB偏构象型特异性单抗的筛选方法,通过对所筛选的单抗进行分析鉴定并建立了CK-MB定量检测试剂,与罗氏试剂检测结果符合率高。     

Characterization of carp thrombocytes with specific monoclonal antibodies

Monoclonal antibodies (WCL6) specific for carp Cyprinus carpio thrombocytes were produced by immunizing mice with membrane lysates of IgM-negative peripheral blood leucocytes (PBL) and selected on the negative reaction with B cells. WCL6 was reactive with a membrane molecule of approximately 90 kDa and to a lesser extent with molecules of approximately 95 and 110 kDa. In general, between 30 and 40% of PBL were WCL6⁺ and appeared to be round to spindle-shaped cells. Immunohistochemical analysis of cryo-sections showed much higher numbers of WCL6⁺ cells in the spleen than in the pronephros, intestine and thymus. Flow cytometric analysis of cell suspensions isolated from these organs only revealed WCL6⁺ cells (4–10%) in the spleen. Electron microscopy of immunogold-stained WCL6⁺ PBL showed round but also some spindle-shaped cells with canalicular and granular structures, and a more irregular and electron-dense nucleus than found in lymphocytes. WCL6⁺ cells with electrondense pyknotic nuclei (without a clear nuclear envelope) were found also and their frequency increased with the length of the isolation and staining procedure used. In the spleen, several differentiation steps of WCL6⁺ cells were found and hence the spleen seems to be the thrombopoietic organ in carp. Thrombocytes from blood could be activated with collagen; the collagen-activated cells showed a higher side (90°) scatter by flow cytometric analysis and finally considerable cell death.     

High-level expression of an endoxylanase gene from Bacillus sp. in Bacillus subtilis DB104 for the production of xylobiose from xylan

To produce xylobiose from xylan, high-level expression of an endoxylanase gene from Bacillus sp. was carried out in Bacillus subtilis DB104. A 1.62-kb SmaI DNA fragment, coding for an endoxylanase of Bacillus sp., was ligated into the Escherichia coli/B. subtilis shuttle vector pJH27Δ88, producing pJHKJ4, which was subsequently transformed into B. subtilis DB104. A maximum endoxylanase activity of 105 U/ml was obtained from the supernatant of B. subtilis DB104 harboring pJHKJ4. The endoxylanase was purified to homogeneity by ion-exchange chromatography and the production profile of xylooligosaccharides from xylan by the endoxylanase was examined by HPLC with a carbohydrate analysis column. Xylobiose was the major product from xylan at 40 °C and its proportion in the xylan hydrolyzates increased with the reaction time; at 12 h, over 60% of the reaction products was xylobiose. These results suggest that xylobiose, which has a stimulatory effect on the selective growth of the intestinal bacterium Bifidobacterium, can be mass-produced effectively by the endoxylanase of Bacillus sp. cloned in B. subtilis. Received: 2 January 1998 / Received revision: 4 March 1998 / Accepted: 4 March 1998