Identification of Bacteroides fragilisby a modified immuno polymerase chain reaction (mIPCR), using a specific monoclonal antibody

Bacteroides fragilis is the anaerobic species most commonly isolated from human clinical specimens, and is resistant to many antimicrobial agents. A monoclonal antibody, mAb4H8 (IgG3), reacting with a specific epitope in the lipopolysaccharide (LPS) isolated from most of the B. fragilis strains, was produced and employed with modified Immuno Polymerase Chain Reaction (mIPCR) for identification of B. fragilis with a detection limit of 10(4) cfu/mL bacterial suspension. A number of bacterial strains were examined, including B. fragilis, Bacteroides spp. other than B. fragilis and other genera. All the B. fragilis strains with the immunodominant (beta1,6-linked D-galactosyl chain) epitope were positive. None of the other strains showed the positive reaction. The results indicate that mIPCR assay with mAb4H8 has a high specificity and high sensitivity.     

Investigation of antigenic expression of Bacteroides fragilis by immunogold labelling and immunoblotting with a monoclonal antibody

Abstract Electron microscopy and immunogold labelling with monoclonal antibody (McAb) Bfl identified an antigen expressed on some in vitro and in vivo grown Bacteroides fragilis NCTC9343 cells.
Immunoprecipitation with this McAb was used to enrich for B. fragilis NCTC9343 cells expressing the Bfl antigen. The McAb Bfl bound to an epitope close to the surface of the outer membrane, but the fibrous capsular network radiating from the bacterial surface was not labelled. Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting identified 3 high- M _r bands which resisted heating and protease digestion but were partially sensitive to sodium periodate treatment.     

16S rRNA PCR鉴定脆弱类杆菌

目的:应用16SrRNA序列设计PCR引物鉴别脆弱类杆菌。方法:通过脆弱类杆菌16SrRNA序列特异性位点设计引物,对4株脆弱类杆菌及大肠杆菌、乳酸杆菌、嗜热链球菌等进行PCR扩增。应用琼脂糖电泳法对PCR扩增产物进行特异性检测。结果:脆弱类杆菌在176bp左右出现特异性条带,而其他细菌均未出现特异性条带。结论:通过16SrRNA序列中特异位点设计引物进行PCR,可特异性鉴定脆弱类杆菌。     

Identification of several forms of the glial fibrillary acidic protein, or alpha-albumin, by a specific monoclonal antibody

Abstract: A monoclonal antibody (mAb), termed UIA/ NEU/I/G1 (G1), that reacted with the astroglial marker glial fibrillary acidic protein (GFAP), or α-albumin, is described. It was directed against a structural determinant of GFAP. The G1 mAb could be used for quantitative determination of GFAP in two-site radiometric assays and for histoimmunological demonstration of GFAP. The G1 mAb reacted with the GFAP from rat as well as from man. The presence of several different molecular weight forms of GFAP in aqueous and detergent extracts from human brain was shown with the G1 mAb. The possible meaning of these forms is discussed.     

Identification of a mouse monoclonal antibody, LHLP-1, specific for human Lp(a)

Heretofore, immunologic reagents used to define and quantify human Lp(a) have been polyclonal in origin and therefore heterogeneous in antigenic specificity. We report here the isolation of a mouse monoclonal antibody, LHLP-1, monospecific for Lp(a). The antigen reactive with LHLP-1 was expressed in both lipoprotein Lp(a) as well as apolipoprotein Lp(a) delipidated by SDS treatment; however, disulfide reduction of apolipoprotein Lp(a) inhibited LHLP-1 reactivity. The antigen reactive with LHLP-1 on Lp(a), therefore, appears not to require lipid for expression of its conformationally dependent (disulfide-inhibitable) epitope. Antigen reactivity was virtually absent in the apoB and other proteins contained in very low density, low density, and high density lipoprotein particles. Immunologic quantification of Lp(a) in individual serum samples with a rabbit reference antiserum or LHLP-1 showed good correlation. We conclude that the monoclonal antibody LHLP-1 identifies an antigen unique to Lp(a) and that this antibody may therefore be useful in the further characterization and measurement of human Lp(a).     

Tryptic Soy Bile-Kanamycin Test for the Identification of Bacteroides fragilis

A simple and practical test for the identification of Bacteroides fragilis is described. It utilizes two well-known properties of this species, i.e., stimulation of growth by bile and resistance to kanamycin. The test media are a tryptic-soy bile agar plate and a supplemented blood agar plate on which a kanamycin 1,000-mug/ml disk is placed. Incubation is for 24 h at 37 C in GasPak. The results of screening 190 strains, mostly clinical isolates, indicate that B. fragilis can be easily and reliably distinguished from other Bacteroides and from Fusobacterium species by its growth on tryptic-soy bile agar and resistance to kanamycin.     

Isolation of a ferritin from Bacteroides fragilis

A ferritin was isolated from the obligate anaerobe Bacteroides fragilis. Estimated molecular masses were 400 kDa for the holomer and 16.7 kDa for the subunits. A 30-residue N-terminal amino acid sequence was determined and found to resemble the sequences of other ferritins (human H-chain ferritin, 43% identity; Escherichia coli gen-165 product, 37% identity) and to a lesser degree, bacterioferritins (E. coli bacterioferritin, 20% identity). The protein stained positively for iron, and incorporated 59Fe when B. fragilis was grown in the presence of [59Fe]citrate. However, the isolated protein contained only about three iron atoms per molecule, and contained no detectable haem. This represents the first isolation of a ferritin protein from bacteria. It may alleviate iron toxicity in the presence of oxygen.     

A Simple Bile-Disk Method for the Identification of Bacteroides fragilis

A simple method for the separation and identification of Bacteroides fragilis from other Bacteroides species through the use of oxgall-impregnated filter paper disks is described. As in the antibiotic sensitivity test, filter paper disks containing defined amounts of bile salt were placed on a lawn of bacterial cells on Gifu anaerobic medium agar plates. Bile-resistant bacteria were identified as strains of Bacteroides fragilis after incubation in anaerobic jars containing hydrogen and carbon dioxide generators at 35 C for 24 hr. The optimum concentration of bile salt, yielding results very similar to those of the conventional tube method, was determined to be 25 mg per disk, empirically. Since this method is easy to perform, time saving, economical and gives clear results, it may be readily used as a daily routine test in the clinical microbiology laboratory.     

Characterization of proteases formed by Bacteroides fragilis

Bacteroides fragilis NCDO 2217 produced three major proteases, P1, P2 and P3 of estimated molecular masses 73, 52 and 34 kDa respectively. Protease P1 weakly hydrolysed azocasein but strongly hydrolysed valyl-alanine p-nitroanilide (VAPNA), glycyl-proline p-nitroanilide (GPRPNA), and to a lesser extent leucine p-nitroanilide (LPNA), indicating it to be an exopeptidase. Proteases P2 and P3 hydrolysed only azocasein and LPNA. The high protease:arylamidase ratios of these enzymes indicated that they were probably endopeptidases. Experiments with protease inhibitors suggested that P1 and P2 had characteristics of serine and metalloproteases respectively and that P3 was a cysteine protease. The proteolytic activity of whole cells was stimulated by divalent metal ions such as Mn2+, Ca2+ and Mg2+, but was strongly inhibited (about 95%) by Cu2+ and Zn2+. The temperature optimum for protein hydrolysis was 43 degrees C. Proteolysis was temperature sensitive, however (90% reduction at 60 degrees C) and was maximal at alkaline pH, with two broad peaks at pH 7.9 and pH 8.8. Cell fractionation showed that P1 was located intracellularly and in the periplasm, whereas P2 and P3 were largely associated with the outer membrane. Release of the membrane-bound proteases by treatment with 1 M-NaCl suggested that ionic interactions were involved in the association of these enzymes with the membranes.     

Haemagglutination of Bacteroides fragilis

Abstract The relationship between the ability to cause haemagglutination (HA) and the presence of capsules and/or pili was examined for 50 clinical isolates of Bacteroides fragilis . HA was tested using a slide technique, and bovine, porcine, guinea pig, rat, rabbit, horse, human, chicken and pigeon erythrocytes. Chicken and pigeon erythrocytes were the best indicators for HA with 43 (86%) of the strains tested causing HA and 39 (78%) with strong reactions. Capsule staining showed that the same 43 strains causing HA also produced a demonstrable capsule. No pili were found on either encapsulated or non-encapsulated strains using transmission electron microscopy. These results suggest that adherence of B. fragilis is related to the presence of capsular material, not pili.     

Analysis of degradation of the basement membrane protein nidogen, using a specific monoclonal antibody

A monoclonal antibody was produced against purified nidogen extracted from a mouse basement-membrane-producing tumor. This antibody reacted with a determinant on Nd-40, a rod which separates the globular domains of nidogen. Antigenicity depends on intrachain disulfide bonds within this rod. The monoclonal antibody was used to detect nidogen fragments after proteolytic cleavage of isolated nidogen, and nidogen complexed to laminin. The data indicate that thrombin and thermolysin generated very different patterns of degradation, but in both cases no differences were found between isolated and complexed nidogen. In contrast, nidogen in the laminin-nidogen complex was much less degraded by trypsin than isolated nidogen, indicating that an interaction between these basement membrane components reduces the susceptibility of nidogen to trypsin digestion. Immunofluorescent studies, using the monoclonal antibody on sections of the EHS tumor after proteolytic digestion, showed that the retention or disappearance of the Nd-40 determinant correlated with the in vitro digestion pattern of the laminin-nidogen complex.     

Entamoeba histolytica: specific antigen recognized by a monoclonal antibody

Specific antigenic determinants on the membrane surface of Entamoeba histolytica that distinguish it from other Entamoeba species were demonstrated. Evidence for these antigenic determinants was obtained with a monoclonal antibody to E. histolytica which showed not only specificity but also sensitivity as demonstrated in enzyme linked immunosorbent assay. Immunofluorescence microscopy showed that the monoclonal antibody recognized an epitope present on the membrane surface of E. histolytica trophozoites. The epitope detected by the monoclonal antibody was present in three components of different molecular weight. These components may have a common precursor or may be the result of enzymatic degradation under the conditions tested.     

Nutritional Features of Bacteroides fragilis subsp. fragilis

Studies of three reference strains of Bacteroides fragilis subsp. fragilis showed that they grow well in a minimal defined medium containing glucose, hemin, vitamin B₁₂, minerals, bicarbonate-carbon dioxide buffer, NH₄Cl, and sulfide. The vitamin B₁₂ requirement of 0.1 ng/ml was replaced with 7.5 μg of methionine. Cysteine or sulfide was an excellent source of sulfur, thioglycolate was a poor source, and thiosulfate, methionine, β-mercaptoethanol, dithiothreitol, sulfate, or sulfite did not serve as sole sources of sulfur. Neither single amino acids, nitrate, urea, nor a complex mixture of L-amino acids or peptides effectively replaced ammonia as the nitrogen source. Comparative studies with a few strains of other subspecies of B. fragilis including B. fragilis subsp. vulgatus, B. fragilis subsp. thetaiotaomicron, and B. fragilis subsp. distasonis indicate that they exhibit similar growth responses in the minimal medium. A single strain of B. fragilis subsp. ovatus required other materials. The results indicate the great biosynthetic ability of these organisms and suggest that, in their ecological niche within the large intestine, many nutrients such as amino acids are in very low supply, whereas materials such as ammonia, heme, and vitamin B₁₂, or related compounds, must be available during much of the time.     

Prevalence of the Bacteroides fragilis Group and Enterotoxigenic Bacteroides fragilis in Immunodeficient Children

Members of the Bacteroides fragilis group are indigenous to the human and animal intestinal microbiota and they are responsible for several endogenous infections. Enterotoxigenic B. fragilis (ETBF) has been associated with acute diarrhea in children and farm animals. Immunodeficient patients are more predisposed to different opportunistic infections, including anaerobic infections. In this study, 130 stool samples were analysed from 56 immunodeficient and 74 healthy children. Enterotoxin production was detected by cytotoxicity assay on HT-29 cells and by PCR. B. fragilis sensu strictu was prevalent in both groups and ETBF species was detected from a single stool sample belonged to an immunodeficient child with AIDS.     

脆弱类杆菌属菌株的培养及免疫血清的制备

本文报告了从国外引进的五种类杆菌培养,鉴定及其抗血清的制备。结果表明这些类杆菌在牛心脑培养基中容易生长。均为厌氧性革兰氏阴性无芽孢多形态杆菌。经生化反应鉴定结果发现脆弱类杆菌对红霉素(60μg)、利福平(15μg)敏感,而对多粘菌素B(10μg)、青霉素(2IU)、卡那霉素(1000μg),万古霉素(5μg)耐受。用不同接种途径免疫家兔所获得的脆弱类杆菌抗血清的结果表明皮下加不完全佐剂组血清中的抗体滴度明显高于一般皮下组及静脉注射组。     

Identification of testis specific calcineurin beta subunit isoform by a monoclonal antibody and detection of a specific six amino acid sequence.

Two isoforms of calcineurin beta subunit(beta 1 and beta 2) were identified in rat testis by a monoclonal antibody Va1. Both beta 1 and beta 2 were recovered in calmodulin binding protein fraction and showed calcium shift on SDS-polyacrylamide gel electrophoresis which is the specific character for EF-hand calcium binding protein. beta 2 showed same apparent molecular weight on SDS-PAGE as that of brain calcineurin beta and was found in wide variety of tissues. beta 1 was shown to have six amino acid polypepeptide sequence and it showed higher molecular weight than brain beta and was specific for testis.     

Identification of three porins in the outer membrane of Bacteroides fragilis

Abstract Four outer membrane proteins were purified to homogeneity from isolated outer membranes of Bacteroides fragilis ; three ( M _r 51000, 92000 and 125 000) had pore-forming activity in reconstituted liposomes as determined by swelling assay. Membrane vesicles containing the M _rmr 55 000 outer membrane protein showed no detectable pore-forming activity. The three B. fragilis porins formed pores that allowed the penetration of uncharged saccharides of M _r lower than 340–400, even though the efficiency of solute diffusion showed slight differences. The diffusion rates of glucose through the porins appeared to be lower than those through Escherichia coli porins.     

Detection of Ki-ras proto-oncogene protein by a specific monoclonal antibody.

We have used a commercially available mouse monoclonal antibody and shown it to bind specifically to cellular Ki-ras p21 proteins and not to cellular N- and Ha-ras p21 proteins. In conjunction with electrophoresis and Western blotting, this antibody can be used, with further detailing, to assess levels of the cellular Ki-ras p21 against a background of total p21s.