Similar binding properties for a neutralizing anti-tetanus toxoid human monoclonal antibody and its bacterially expressed Fab.
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Amino Acid Sequence↗
Biomedical subjects
F Nato
Publications and source records attributed to F Nato.
Internalin must be on the bacterial surface to mediate entry of Listeria monocytogenes into epithelial cells.
Entry of Listeria monocytogenes into cultured epithelial cells requires production of internalin, a protein with features characteristic of some Gram-positive bacterial surface proteins, in particular an LPXTG motif preceding a hydrophobic sequence and a few basic residues at its C-terminal end. By immunofluorescence and immunogold labelling, we show that in wild-type L. monocytogenes, internalin is present on the cell surface and has a polarized distribution similar to that of ActA, another surface protein of L. monocytogenes involved in actin assembly. Through a genetic analysis, we establish that the C-terminal region of internalin is necessary for cell-surface association, and that although internalin is partially released in the culture medium, its location on the bacterial surface is required to promote entry. Finally, using a 'domain-swapping' strategy-replacement of the cell wall anchor of IniA by the membrane anchor of ActA- we show that the reduced ability to adhere and enter cells of strains expressing IniA-ActA correlates with a lower amount of surface-exposed internalin. Taken together, these results suggest that internalin exposed on the bacterial surface mediates direct contact between the bacterium and the host cell.
Amino Acid Sequence↗
Antibodies to the leucine-rich repeat region of internalin block entry of Listeria monocytogenes into cells expressing E-cadherin.
Internalin, a surface protein essential for entry of Listeria monocytogenes EGD into epithelial cells, was used as an antigen to raise nine monoclonal antibodies. These monoclonal antibodies recognized seven distinct epitopes which were located in three different regions of the protein. Three of them inhibited internalin-mediated entry and recognized the amino-terminal leucine-rich repeat region of the protein, suggesting that this region is essential for entry.
Animals↗
Lack of cleavage of IcsA in Shigella flexneri causes aberrant movement and allows demonstration of a cross-reactive eukaryotic protein.
Once in the cytoplasm of mammalian cells, Shigella flexneri expresses a motile phenotype caused by polar directional assembly of actin. This process depends on accumulation of IcsA (VirG), a 120-kDa protein with ATPase activity, at the pole of the bacterium opposite to that at which ongoing septation occurs. IcsA is also secreted into the bacterial supernatant as a 95-kDa species, after cleavage at an SSRRASS sequence which, when mutagenized, blocks processing. MAbF15, an anti-IcsA monoclonal antibody, recognizes an epitope located within repeated Gly-rich boxes in the N-terminal half of the protein. We used this monoclonal antibody to visualize the location of a noncleavable 120-kDa IcsA mutant protein expressed in S. flexneri. We found that this noncleavable IcsA protein no longer localized exclusively to the pole of the bacterium but also could be detected circumferentially. Whereas the monoclonal antibody detected the wild-type cleavable form of IcsA in only 40% of the cells expressing this protein, the noncleavable was easily detectable in all the cells carrying the icsA mutant allele. Similar aberrant localization of the IcsA mutant protein on bacteria growing within the cytoplasm of HeLa cells was observed. The strains expressing the noncleavable IcsA protein expressed abnormal intracellular movement and were often observed moving in a direction perpendicular to their longitudinal axis. The putative protease which processes IcsA may therefore play a role in achieving polar expression of this protein and providing maximum asymmetry essential to directional movement. In addition, MAbF15 allowed us to identify a 70-kDa eukaryotic protein cross-reacting with IcsA. This protein accumulated in the actin tails of motile bacteria and in membrane ruffles of the cells.
Actins↗
Development of a bispecific monoclonal antibody for use in molecular hybridisation.
A mouse hybrid hybridoma (tetradoma) was prepared by fusing hybridomas producing monoclonal antibody to acetyl-aminofluorene with hybridomas producing antibody against calf intestine alkaline phosphatase. The tetradoma line established secreted immunoglobulin manifesting parental and bispecific binding characteristics. Bispecific monoclonal antibody was purified and used for a one-step immunodetection assay of non-radioactive DNA and RNA probes. The immunoassay developed was able to detect 5 pg DNA within 2 h and gave low background noise.
2-Acetylaminofluorene↗
Comparison of various methods and reagents for species identification of Staphylococcus aureus positive or negative for the mecA gene.
The reliability of various methods for species identification of Staphylococcus aureus was evaluated. A total of 135 coagulase-positive (SA) or -negative (SS) staphylococcal isolates were tested, including methicillin-resistant (MR) and -susceptible (MS) strains. When the nuc gene which encodes the S. aureus thermonuclease (TNase) was amplified in a multiplex PCR simultaneously with the mecA gene which encodes for the MR-associated penicillin-binding protein 2a of staphylococci, the nuc amplification showed full agreement with the results of the coagulase test. TNase detected by an enzymatic method or as protein in a sandwich ELISA identified S. aureus with nearly the same precision as the PCR. The Staphylase, Monostaph and Staphaurex agglutination kits were all reliable for identification of MSSA, but not for MRSA. Most of the negative MRSA strains were identified by the Pastorex agglutination kit, in which reagents for fibrinogen receptor and protein A detection have been supplemented with antibodies for capsular polysaccharides of the serotypes 5 and 8. These results show that detection of the nuc gene or its TNase product is highly reliable for identification of both MRSA and MSSA strains, while various widely used agglutination kits do not show the same reliability for identification of MRSA strains.
Agglutination Tests↗
Characterization of B-cell epitopes on IpaB, an invasion-associated antigen of Shigella flexneri: identification of an immunodominant domain recognized during natural infection.
The invasion plasmid antigen B (IpaB), a 62-kDa plasmid-encoded protein associated with the ability of shigellae to invade epithelial cells, is the bacterial antigen most strongly and consistently recognized by the host during infection. The strong systemic and mucosal immune responses observed against this invasin prompted us to map its B-cell epitopes. For this purpose, IpaB was first overexpressed in Shigella flexneri and used to raise rabbit polyclonal antiserum and murine monoclonal antibodies, which were subsequently used to screen a lambda gt11 ipaB library. Inserts of recombinant DNA clones that were specifically recognized by the antisera and antibodies were sequenced, and three distinct determinants were identified. Further characterization of these determinants showed that they were recognized by sera from patients convalescent from shigellosis, suggesting that they are relevant to the humoral response during natural infection. Moreover, the IpaB region comprising the three determinants was systematically recognized by all sera from infected patients that we tested, whereas other regions of the protein were not. These data suggest that this region, located between amino acid residues 147 and 258, is the major immunogenic domain of the invasin in the course of natural infection.
Animals↗
New latex reagent using monoclonal antibodies to capsular polysaccharide for reliable identification of both oxacillin-susceptible and oxacillin-resistant Staphylococcus aureus.
A new latex agglutination test (Pastorex Staph-Plus, Sanofi Diagnostics Pasteur), consisting of a mixture of latex particles coated with fibrinogen and immunoglobulin G for the detection of clumping factor and protein A and latex particles sensitized with monoclonal antibodies directed to Staphylococcus aureus serotype 5 and 8 capsular polysaccharides, was compared with three commercially available rapid agglutination methods for the identification of 220 isolates of S. aureus (61 oxacillin resistant) and 128 isolates of coagulase-negative staphylococci. The sensitivity for identification of S. aureus was high with the Pastorex Staph-Plus test (98.6%) compared with those of the other tests, which ranged from 91.8 to 84.5%. Test sensitivities for the identification of oxacillin-resistant S. aureus were as follows: Pastorex Staph-Plus, 95.1%; Pastorex Staph, 73.8%; Staphyslide, 72.1%; and StaphAurex, 49.2%.
Antibodies, Monoclonal↗
Monoclonal antibodies for identification of Borrelia afzelii sp. nov. associated with late cutaneous manifestations of Lyme borreliosis.
Borrelia isolates associated with Lyme borreliosis were previously divided into 3 genospecies, B. burgdorferi sensu stricto, B. garinii and group VS461, on the basis of DNA homology. B. burgdorferi sensu stricto and B. garinii were identified by monoclonal antibodies (MAbs), H3TS and D6 respectively, but no MAbs were available to identify group VS461. Two MAbs were produced, I 17.3 and J 8.3 which reacted with OspB and OspA proteins, respectively, of strains belonging to group VS461, which should be named B. afzelii sp. nov. 24 strains were assigned to B. afzelii sp. nov., 11 of them being isolated from skin lesions, 6 from acrodermatitis chronica atrophicans (ACA) and 5 from erythema chronicum migrans (ECM). Although quite unknown in the USA, ACA has frequently been reported in northern Europe where B. afzelii sp. nov. is commonly isolated. This study documents the involvement of B. afzelii sp. nov. as a specific aetiological agent of ACA.
Acrodermatitis↗
Identification and characterization of B-cell epitopes of IpaC, an invasion-associated protein of Shigella flexneri.
Invasion plasmid antigen C (IpaC) is a 43-kDa plasmid-encoded protein associated with the ability of shigellae to invade epithelial cells. This protein is consistently strongly recognized by sera from convalescent patients and monkeys experimentally infected with shigellae. The strong immunogenicity of IpaC in the course of natural infection makes it a good candidate as a potentially protective antigen. To map the B-cell epitopes of this protein, the gene encoding IpaC was cloned and expressed at a high level in Escherichia coli. The partially purified recombinant protein was used to raise rabbit polyclonal antisera and murine monoclonal antibodies. A lambda gt11 ipaC gene library was screened with the antisera and antibodies. Recombinant DNA clones producing specific antigenic determinants were isolated, and the sequence of their DNA inserts was determined. The amino acid sequence of each determinant was deduced from the minimal overlap of DNA inserts of multiple antibody-positive DNA clones. Two distinct epitopes, located between amino acid residues 25 and 33 and 90 and 97, were identified. Two additional B-cell epitopes which were located between residues 297 and 349, near the carboxy-terminal end of the protein, were characterized. Each of these epitopes was also recognized by sera from convalescent humans and monkeys. Therefore, it seems likely that these epitopes are relevant to the humoral response against IpaC during natural infection.
Animals↗
Detection of Pneumocystis carinii in bronchoalveolar lavage fluid by dot immunobinding assay and immunofluorescence.
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AIDS-Related Opportunistic Infections↗
Production and characterization of monoclonal antibodies to human Pneumocystis carinii for the diagnosis of P. carinii pneumonia.
OBJECTIVES: Monoclonal antibodies against human Pneumocystis carinii were produced by fusion of myeloma cells (X63-AG8.653) with splenocytes from Biozzi mice that had been immunized against P. carinii cysts isolated from infected human lung. The aim of this study was to evaluate the usefulness of these monoclonal antibodies for the diagnosis of P. carinii pneumonia by indirect immunofluorescence in comparison with a modified silver stain method and commercial kits. METHODS: One hundred fifty-seven specimens from 87 patients, infected or non-infected with human immunodeficiency virus, were examined for the presence of P. carinii. Specimens were either induced sputum samples or bronchoalveolar lavage fluids. Indirect immunofluorescence was performed with six stable clones obtained by limiting dilution. Four of the monoclonal antibodies were IgG1 isotypes, one was an IgG3 and one was an IgM. Their isoelectric points varied from 6.5 to 8.3. Tests were also performed with silver methenamine staining and with two commercially available monoclonal antibodies (Monofluo kit from Diagnostics Pasteur and MAb from Dako). RESULTS: The 6 antibodies all recognized P. carinii cysts in indirect immunofluorescence. No cross reactivity was observed with yeast or host cells. P. carinii antigens could not be identified with western immunoblotting suggesting that the monoclonal antibodies recognized native antigens. This result was confirmed by dot blot analysis. Spots were observed with native but not with denatured antigens. Inhibition studies showed that these 6 antibodies recognized the same or overlapping sites. The sensitivities of detection of P. carinii in sputum were 87% by silver stain and from 93.5 to 96.7% by immunofluorescence. The sensitivities of detection in bronchoalveolar lavage were 67.3% by silver stain and from 75.7% to 76.8% by immunofluorescence. CONCLUSION: Immunofluorescence was more sensitive than silver staining and the best results were obtained with E5-8 and A8-13 monoclonal antibodies and with Monofluo kit.
Animals↗
Immunological screening of a glycoprotein antigen expressed by Zajdela ascites hepatoma cells on normal rat tissues and tumour cells.
Expression of the glycoprotein MII2 antigen originally identified in Zajdela ascites hepatoma cells was investigated in several normal rat tissues and in more or less differentiated tumours using biochemical and immunological approaches. SDS-polyacrylamide gel electrophoresis followed by fluorography or immunoblotting with an antiserum raised against the purified MII2 antigen revealed that this antigen was absent from normal liver cells. ELISA assays, indirect immunofluorescence and immunoprecipitation experiments using the same antiserum showed that this glycoprotein was not expressed in various normal tissues such as liver, spleen, lung, pancreas, intestine and stomach, but it was unexpectedly detected in kidney and thymic tissues. However, the molecular weight of the antigens immunoprecipitated from kidney and thymus was lower than the one of MII2 (Mr of 60,000 versus 110,000-160,000 for purified MII2). No staining was observed in embryonic rat liver at 10 and 20 days of development. Moreover, this antigen was present on the surface of Morris hepatoma 7777, another rapidly proliferating and poorly differentiated hepatocellular carcinoma. In contrast, this antigen was not detected on the surface of in vitro Zajdela hepatoma cells (ZHC) or of partially differentiated hepatomas (Faza) which have recovered some hepatic functions. In addition, the MII2 antigen was found on the human non-hepatic HT-29 tumour cell line, under its undifferentiated form (HT-29 G+ subline). The possible relationships between the expression of this antigen and both the malignant transformation process and the differentiation process are discussed.
Animals↗
Production and characterization of neutralizing and nonneutralizing monoclonal antibodies against listeriolysin O.
Listeriolysin O (LLO) is a thiol-activated toxin secreted by the facultative intracellular pathogen Listeria monocytogenes. LLO is essential for the survival of the bacterium in the infected cell because it promotes lysis of the phagosome membrane and escape of the bacterium into the cytosol. LLO was used as an antigen for the production of nine monoclonal antibodies (MAbs) in mice. Three of these could inhibit the hemolytic activity of LLO. One of them inhibited binding of LLO to erythrocyte membranes. The two other antibodies blocked the activity of LLO at a step subsequent to membrane binding. Only two of the nine MAbs recognized three other purified SH-activated toxins, streptolysin O, alveolysin, and pneumolysin. Western blot (immunoblot) analysis of culture supernatants of Listeria ivanovii and Listeria seeligeri, two hemolytic species of the genus Listeria, revealed that two MAbs recognized ivanolysin and seeligerolysin. The latter was also recognized by two other MAbs, including one of the neutralizing antibodies. MAbs raised against a peptide, ECTG LAWEWWR, present in all thiol-activated toxins sequenced to date, recognized all toxins and were not neutralizing. Taken together, these results demonstrate the existence of regions important for hemolytic activity that are unique to hemolysins of the genus Listeria and show that regions outside the conserved peptide are important for activity of LLO.
Amino Acid Sequence↗
Production of polyclonal and monoclonal antibodies against group A, B, and C capsular polysaccharides of Neisseria meningitidis and preparation of latex reagents.
Polyclonal and monoclonal antibodies against capsular polysaccharides of Neisseria meningitidis serogroups A, B, and C were produced in order to develop immunological reagents allowing both the detection of soluble antigens during meningococcal meningitis and antigenic serogrouping of N. meningitidis cultures. The performance characteristics of monoclonal and polyclonal antibody latex reagents were compared. For the detection of soluble polysaccharide antigen, polyclonal antibody latex reagent was selected for N. meningitidis A and C. The latex reagent prepared with polyclonal antibodies against N. meningitidis B could not detect capsular polysaccharide even at 1 mg/ml. The monoclonal antibody B latex reagent which detected 100 ng of polysaccharide per ml was therefore chosen. For the serogroup identification of N. meningitidis, the use of a confirmatory test results in an overall specificity of 100% with polyclonal or monoclonal antibody latex reagents.
Antibodies, Bacterial↗
Biosynthesis of high molecular weight polylactosamine-type glycopeptides in rat Zajdela hepatoma ascites cells.
The first steps of the biosynthetic pathway of high molecular weight polylactosamine-type glycopeptides from rat Zajdela hepatoma cells were studied by pulse-chase experiments, biochemical analysis and by inhibition of N-glycosylation. It is clear that this process involves firstly the transfer of a lipid-linked high-mannose oligosaccharide precursor to a protein moiety in a similar way to that of N-linked glycopeptides of a more common size range according to the classical 'cycle of dolichol'. In the presence of enzymes which are inhibitors of the processing of high-mannose oligosaccharide chains, this class of oligosaccharides was considerably increased, whereas polylactosamine chains and lower complex N-linked glycopeptides were concomitantly decreased in the same kinetics and the same ratio. As expected in the presence of N-methyldeoxynojirimycin, which is an alpha-glucosidase inhibitor, high-mannose oligosaccharides remained glycosylated and are mostly of the Glc1-3Man9GlcNAc type. In the presence of swainsonine, which is an alpha-mannosidase (EC 3.2.1.24) inhibitor, these chains were devoid of glucose residues. In addition, some chains displayed hybrid structures. It appears, therefore, that the first steps of the biosynthesis of polylactosamine-type and N-linked oligosaccharides of a more common size range proceed similarly and that differences between their biosynthetic pathways occur during the elongation phase, which leads to their final respective structures. Glycopeptides prepared from the cell surface by mild trypsin treatment as well as from entire cells, previously treated or not by processing inhibitors, display the same gel filtration patterns indicating that modifications in protein glycosylation do not prevent glycoprotein insertion into the cell membrane.
Amino Sugars↗
Expression of alpha and beta tropomyosin subunits during early myogenesis in somites and limb buds of chick embryos.
The appearance of alpha and beta subunits of skeletal tropomyosin in early myogenesis was studied histochemically using monoclonal antibody to alpha tropomyosin and affinity-purified polyclonal antibody to beta tropomyosin. In muscle cells, in both somites and limb buds, the alpha and beta subunits are simultaneously expressed and first appear in the somites at the 30-36 somites. The relatively greater amount of beta than alpha tropomyosin found in early myogenesis is thus likely to result from a higher rate of beta tropomyosin synthesis.
Animals↗
Direct testing of blood culture for detection of the serotype 5 and 8 capsular polysaccharides of Staphylococcus aureus.
Monoclonal antibodies (MAbs) reactive with serotype 5 and 8 capsular polysaccharides of Staphylococcus aureus have been used to test, by enzyme-linked immunosorbent assay (ELISA), blood culture fluids for the presence of S. aureus. A total of 748 blood cultures from 665 patients yielding 706 bacterial isolates belonging to more than 26 bacterial species were studied. All blood cultures containing bacterial strains belonging to species other than S. aureus were negative in ELISA. All 23 blood cultures containing serotype 5 S. aureus were positive in ELISA with the corresponding MAb. Out of 20 blood cultures containing serotype 8 S. aureus, 19 were positive with the corresponding MAb. All 5 blood cultures containing nontypeable S. aureus were negative in ELISA with both MAbs. This method provides reliable identification of serotype 5 or serotype 8 S. aureus by direct testing of blood culture fluids with ELISA.
Animals↗