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J M Luk

Publications and source records attributed to J M Luk.

33 records · Page 2Linked to original sources
PubMed1997

Signaling mechanisms of pertussis toxin-induced myelomonocytic cell adhesion: role of tyrosine phosphorylation.

Pertussis toxin (PTX) was thought to bind Mac-1 integrin receptor (CD11b/CD18) on TGF-beta1/D3-primed U937 cells and induced cellular adhesion to serum-coated plate. The present study was to investigate the signal transduction pathway utilized by PTX to initiate myeloid cell adhesion in serum. Immunoblotting study showed that PTX induced tyrosine phosphorylation of two cytoplasmic proteins of 150 kDa and 90 kDa in TGF-beta1/D3-primed U937 cells in a time-dependent manner. In addition, PTX-induced myelomonocytic cell adhesion was abolished in the presence of genistein (100 microM), a specific tyrosine kinase inhibitor. 2LPM19c (2 microg/ml), a mouse monoclonal antibody against the CD11b subunit of Mac-1 integrin, or ethylenediamine tetraacetic acid (EDTA, 5 mM) prevented PTX-mediated U937 cell adhesion. On the other hand, nifedipine (1 microM), a calcium channel blocker, significantly reduced PTX-induced U937 cell adhesion. Taken together, it is suggested that binding of PTX to Mac-1 integrin receptor on primed U937 cells triggers protein tyrosine phosphorylation and, to a lesser extent, Ca(+2) influx, which eventually lead to monocytic cell adhesion in serum.

Calcitriol↗
PubMed1997

An enzyme-linked immunosorbent assay to detect PCR products of the rfbS gene from serogroup D salmonellae: a rapid screening prototype.

We describe a digoxigenin-based enzyme-linked immunosorbent assay (DIG-ELISA) following a PCR to detect the amplified lipopolysaccharide rfbS gene as a means for rapid screening of serogroup D salmonellae in stool specimens. For pure bacterial cultures, the sensitivity of the PCR DIG-ELISA was approximately 10 bacteria. In the presence of stool materials, the salmonellae were first isolated by an immunomagnetic separation technique with an O9-specific monoclonal antibody. MATy-O9, followed by PCR and DIG-ELISA. The corresponding sensitivity was about 10 to 100 bacteria. To evaluate the assay performance clinically, 203 stool samples from patients with diarrhea were subjected to the routine culture techniques and the PCR ELISA method with overnight enrichment. The conventional culture method identified 145 salmonellae (31 serogroup B, 27 serogroup C, 83 serogroup D, and 5 serogroup E isolates) and 58 non-salmonella bacteria. The PCR ELISA method correctly identified all 82 serogroup D salmonellae (A405 by ELISA, 2.54 +/- 0.74) but was negative for the other Salmonella serogroups (A405, 0.26 +/- 0.08; n = 63) and non-Salmonella isolates (A405, 0.16 +/- 0.04; n = 58). In order to obtain a visible result, the assay takes approximately 6 h (PCR, 4 h; ELISA, 2 h), along with brief enrichment cultivation of the samples (from 4 to 16 h). Thus, the PCR DIG-ELISA offers a fast, accurate, semiquantitative means of detecting infectious agents such as salmonellae, and future robotic automation is possible.

Bacteriological Techniques↗
PubMed1996

Immunochemical characterization of a haemagglutinating antigen of Arcobacter spp.

The Arcobacter haemagglutinin has been identified by Western immunoblot to be an immunogenic protein of about 20 kDa. The haemagglutinating activity is sensitive to proteolytic enzyme digestion and heat treatment of 80 degrees C and above. The Arcobacter haemagglutinin is possibly a lectin-like molecule binding to erythrocytes via a glycan receptor containing D-galactose as part of its structure.

Animals↗
PubMed1995

Biotinylated lipopolysaccharide binds to endotoxin receptor in endothelial and monocytic cells.

Endotoxin or lipopolysaccharide (LPS), a major cell surface component of gram-negative bacteria, which could bind to different cell types when released into the bloodstream, plays a central role in the pathogenesis of septic shock syndromes. We have established a biotinylation procedure for labeling purified LPS molecules from Salmonella minnesota R595 and Escherichia coli J5 bacteria. The biotin group was conjugated to the bacterial LPS either by chemical oxidation of the LPS carbohydrate moiety (inner core region), followed by reduction with biotin-LC-hydrazide (biotinamido hexanoyl hydrazide), or by photoactivatable cross-linking with biotin-LC-ASA [1-(4-azidosalicylamido-)-6(biotinamido)-hexane], which was randomly attached to the carbohydrate and fatty acid (lipid A) groups of the LPS. Both labeled products retained biological activity (or endotoxicity) as evidenced by coagulation of the Limulus amoebocyte lysate. To determine its ability to bind avidin/streptavidin which in turn could be conjugated with enzymatic and fluorescent probes, the biotinylated LPS was used in enzyme immunoassay, Western blot, and flow cytometry. These assays were also used to analyze the binding of LPS ligand to its counterreceptor(s) on whole cell surface, membrane fragments, and in detergent lysates from human endothelial and monocytic cells. The described biotinylated LPS probes can be applied in a wide variety of techniques in receptor biochemistry, immunohistochemistry, and molecular cell biology.

Avidin↗
PubMed1994

Comparison of three stool-processing methods for detection of Salmonella serogroups B, C2, and D by PCR.

Three different stool sample-processing methods (centrifugation, immunomagnetic separation, and selective enrichment cultivation) for the identification of Salmonella serogroups by PCR were studied. The corresponding sensitivities in an ethidium bromide stained-agarose gel were 10(5), 10(3), and 10 bacteria, respectively. The PCR assay with overnight enrichment performed as well as, or even better than, the conventional culture technique. Of 485 clinical stool samples, PCR correctly identified all 230 culture-positive samples as well as mixed Salmonella infections in four cases.

Bacteriological Techniques↗
PubMed1993

Selective amplification of abequose and paratose synthase genes (rfb) by polymerase chain reaction for identification of Salmonella major serogroups (A, B, C2, and D).

Many parts of the Salmonella rfb gene clusters which are responsible for biosynthesis of the oligosaccharide-repeating units of the O-antigenic lipopolysaccharide have recently been cloned and sequenced. On the basis of this knowledge, three sets of nucleotide primers were selected to target defined regions of the abequose and paratose synthase genes: rfbJ of Salmonella serogroup B, rfbJ of Salmonella serogroup C2, and rfbS of Salmonella serogroup D (also present in serogroup A). For good differentiation among these major serogroups, the primers were designed not only to give precise specificity in priming but also to give DNA products with different sizes in polymerase chain reactions (product sizes, approximately 720 bp for both serogroups A and D, approximately 820 bp for serogroup C2, and approximately 882 bp for serogroup B). In a polymerase chain reaction assay utilizing these rfb-specific primers, all of the 40 salmonellae belonging to serogroups B, C2, and D plus A were accurately identified among a total of 123 clinical isolates tested (including 55 salmonellae from 36 different serotypes and 68 strains from 10 other members of the family Enterobacteriaceae). No false-positive reactions were detected. The selected rfb gene sequences were proved for the first time to be useful DNA-based markers for identification of and differentiation among Salmonella serogroups A, B, C2, and D.

Base Sequence↗
PubMed1992

Specificity of monoclonal antibodies binding to the polysaccharide antigens (Vi, O9) of Salmonella typhi.

A panel of monoclonal antibodies were generated against the surface polysaccharide antigens of the cell envelope of Salmonella typhi. Four clones (IgM) were specific for the capsular Vi polysaccharide, and one clone (IgG3) reacted selectively with the S. typhi lipopolysaccharide in enzyme immunoassay. On the basis of their reactivity pattern and binding affinity, MATy-V7 (IgM) and MATy-O9 (IgG3) antibodies were selected for further characterization of their antigenic specificity. In an inhibition enzyme immunoassay with rabbit factor-specific anti-Salmonella antibodies as the competing agents, the reactivity of MATy-V7 and MATy-O9 were significantly inhibited by the anti-Vi and anti-O9 antisera, respectively. Moreover, both the Vi- and O9-specific monoclonal antibodies were shown to be useful serotyping agents by correct identification in slide agglutination tests of 32 clinical isolates of all the S. typhi and other serogroup D salmonellae among a total of 140 bacterial isolates representing eight different Enterobacteriaceae genera tested.

Antibodies, Monoclonal↗
PubMed1991

Epitope mapping of four monoclonal antibodies recognizing the hexose core domain of Salmonella lipopolysaccharide.

Four murine monoclonal antibodies reactive with distinctive regions of the hexose core domain of Salmonella lipopolysaccharide (LPS) were generated and their epitope specificities were delineated. MAST 56 (IgG1) and MAST 50 (IgG3) antibodies elicited by immunizations with Salmonella typhimurium Rb1 and Rb2 mutants, reacted selectively in enzyme immunoassay with the LPS from rough mutants. In contrast, MATy 1 (IgM) and MATy 2 (IgG2b) antibodies raised by an attenuated Salmonella typhi 620 Ty strain were reactive with LPS from both smooth and rough Salmonellae. Immunoblotting analysis showed that MATy 1 distinguished only the bottom bands (naked LPS core) among the heterogeneous LPS populations, whereas MATy 2 gave a ladder pattern (reactive with both naked and O-chain-substituted LPS cores). Differential binding specificities of MATy 1 and MATy 2 antibodies to the naked and capped LPS cores were further analyzed utilizing S. typhimurium polysaccharide fractions with different O-chain:core ratios which were obtained after separation by Sephacryl S-200 chromatography. Steric effects on the antibody reactivity by the bulky O-polysaccharide chain were detected. The use of chemically defined native and synthetic saccharides as inhibitors, in combination with the conformation of the Salmonella core oligosaccharide, permitted the definition of antigenic determinants carried in the core domain recognized by each antibody: (i) the branches I and VIII are essential for MATy 1 recognition, (ii) the backbone III-IV-V for MATy 2, (iii) the backbone II-III-IV-V for MAST 56, and (iv) the backbone plus the branch III-IV-V-VIII for MAST 50. (formula; see text)

Antibodies, Bacterial↗
PubMed1991

Rapid and sensitive detection of Salmonella (O:6,7) by immunomagnetic monoclonal antibody-based assays.

An immunomagnetic technique to detect and identify Salmonella serogroup C1 has been developed. Monoclonal antibodies (MAbs) specific for the O antigen 6,7 of Salmonella lipopolysaccharide (LPS) coupled to magnetic beads were used to isolate the salmonellae. Captured bacteria were easily identified by acridine orange staining and measured by enzyme immunoassays with a conjugate anti-LPS MAb as the detector probe. The whole detection process required 2-3 h and the sensitivity was 10(3)-10(4) bacteria/ml. The presence of blood (10%, v/v) or stool (1%, w/v) components did not interfere with the immunomagnetic assay performance.

Animals↗
PubMed1991

Anti-Salmonella lipopolysaccharide monoclonal antibodies: characterization of Salmonella BO-, CO-, DO-, and EO-specific clones and their diagnostic usefulness.

To facilitate the identification and serotyping of Salmonella species, we established a wide variety of murine monoclonal antibodies (MAbs) that were reactive with the lipopolysaccharides (LPSs) of Salmonella serogroups B to E. An effective approach for generating LPS-reactive hybridomas was used; this required immunization of mice with LPS-coated bacteria. To screen for diagnostically useful MAbs, the MAbs were tested by enzyme-linked immunosorbent assay against a set of purified LPSs from smooth and rough Salmonella strains. At least four major groups of antibody specificities were identified: Salmonella (i) BO specific, (ii) CO specific, (iii) DO specific, and (iv) EO specific. For a more detailed epitope analysis, a panel of eight different serogroup-specific MAbs which were shown to bind the O-antigenic polysaccharide chains, yielding characteristic ladder patterns in Western blots (immunoblots) against the LPS of Salmonella serogroups B to E, were selected. The availability of various chemically defined LPS structures and Salmonella O-antigen glycoconjugates permitted the definition of O-antigenic polysaccharide epitopes recognized by each MAb that serologically corresponded to factors O3, O4, O5, O6, O7, O8, O9, and O10 on the basis of the Kauffmann-White scheme for Salmonella classification. The diagnostic accuracy of these immunochemically defined O-specific MAbs for Salmonella serotyping was demonstrated by correct identification of all 167 salmonellae (including 72 serotypes from serogroups B to E) among the 294 bacterial strains in a slide agglutination test. No false-positive reactions were detected.

Animals↗
PubMed1990

Efficient production of mouse and rat monoclonal antibodies against the O antigens of Salmonella serogroup C1, using LPS-coated bacteria as immunogen.

A variety of different immunogens and immunisation schemes were investigated for the production of monoclonal antibodies directed against the O antigenic polysaccharide of the lipopolysaccharide (LPS) of Salmonella serogroup C1 (O:6,7). Of 12 fusions performed, higher yields of stable, LPS-reactive hybridomas producing monoclonal antibodies were achieved in both the mouse and rat when using O:6,7 LPS-coated S. thompson bacteria as immunogens than with live and heat-killed bacteria, or O:6,7-BSA glycoconjugate as immunogens. All of the 17 hybrid clones obtained were shown to bind the O antigens of Salmonella serogroup C1 when tested in ELISA against a set of chemically defined LPS from Salmonella smooth and rough strains. The results are discussed with a view to bettering the immunisation strategy for production of monoclonal antibodies against the LPS antigens of bacteria.

Animals↗
PubMed1989

Detection in rabbit sera of blocking antibodies against staphylococcal fibronectin-binding protein by enzyme-linked immunosorbent assay.

Antibody titres against fibronectin-binding protein (FnBP) of Staphylococcus aureus were determined in sera from rabbits immunized with staphylococcal whole cells or purified native fibronectin receptor. An ELISA technique for detection of antibody titres blocking the binding of soluble fibronectin to immobilized FnBP was developed. A recombinant staphylococcal FnBP fused to E. coli beta-galactosidase (gal-FnBp) was used as the immobilized antigen in this test. Serum samples from two different rabbits immunized with native fibronectin receptor gave significant blocking titres, whereas the blocking titres of antisera against staphylococcal whole cells were about 4- to 5-fold lower. Using the gal-FnBP fusion protein, the sensitivity for detection of fibronectin by ELISA was also determined. The detection limit is around 5 ng. The findings are discussed with a view to developing an anti-staphylococcal adherence vaccine and quantitating fibronectin in solution.

Animals↗
PubMed1988

Characterisation and application of a murine monoclonal antibody specific for the serogroup C2 Salmonella.

An IgG3 murine monoclonal antibody (designated MO8) specific for the serogroup C2 Salmonella lipopolysaccharide (LPS) was generated by fusing mouse myeloma cells NS1 with spleen cells of BALB/c mice immunised with heat-killed S. manhattan. MO8 reacted with purified LPS prepared from serogroup C2 Salmonella but did not react with that prepared from other O serogroups, and its reactivity was also specifically absorbed by serogroup C2 Salmonella only. Polyacrylamide gel electrophoresis of the serogroup C2 LPS and subsequent immunoblotting with MO8 yielded multiple reactive bands giving a characteristic ladder pattern. The specificity of MO8 was further demonstrated in the slide agglutination test with 223 bacteria, of which only 25 belonging to serogroup C2 Salmonella reacted with the MO8 ascitic fluid. The specificity of MO8 makes it useful not only for the serological identification of Salmonella but also for the epitope analysis of the serogroup C2 LPS.

Agglutination Tests↗