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M K Gentry

Publications and source records attributed to M K Gentry.

At least 37 records · Page 2Linked to original sources
PubMed1990

Immunocytochemical localization of phosphatidylinositol-anchored acetylcholinesterase in excitable membranes of Torpedo ocellata.

In Torpedo electric organ much of the acetylcholinesterase is a 'globular' dimer (G2), anchored to the plasma membrane via covalently attached phosphatidylinositol and solubilized by a bacterial phosphatidylinositol-specific phospholipase C. This suggested that selective solubilization with phosphatidylinositol-specific phospholipase C, coupled with immunocytochemistry, might be used to localize G2 acetylcholinesterase in excitable tissues of Torpedo. Cryostat sections of electric organ, electromotor nerve, electric lobe and back muscle from Torpedo ocellata were labelled, using three different antibody preparations to Torpedo acetylcholinesterase, followed by a fluorescent second antibody, before and after exposure to the phospholipase. Sites of innervation on electrocytes and myofibers were labelled selectively, as were motor and electromotor nerves. In all these cases labelling was substantially diminished by prior exposure to the phospholipase. The results support our previous assignment, based on biochemical evidence, for a neuronal and synaptic localization of the G2 acetylcholinesterase in Torpedo. Electric lobe acetylcholinesterase appears insensitive to the phospholipase treatment and lacks certain epitopes present in both electric organ and electromotor nerve enzyme. This suggests that substantial processing of the G2 form occurs concomitantly with its movement from the electric lobe into the electromotor nerve.

Acetylcholinesterase↗
PubMed1990

Studies on the topography of the catalytic site of acetylcholinesterase using polyclonal and monoclonal antibodies.

Polyclonal and monoclonal antibodies were generated against a synthetic peptide (25 amino acid residues) corresponding to the amino acid sequence surrounding the active site serine of Torpedo californica acetylcholinesterase (AChE). Prior to immunization, the peptide was either coupled to bovine serum albumin or encapsulated into liposomes containing lipid A as an adjuvant. To determine whether this region of AChE is located on the surface of the enzyme and thus accessible for binding to antibodies, or located in a pocket and thus not accessible to antibodies, the immunoreactivity of the antibodies was determined using enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, Western blots, and competition ELISA. The polyclonal antibody and several of the monoclonal antibodies failed to react with either Torpedo or fetal bovine serum AChE in their native conformations, but showed significant cross-reactivity with the denatured enzymes. Human serum butyrylcholinesterase, which has a high degree of amino acid sequence homology with these AChEs, failed to react with the same antibodies in either native form or denatured form. Chymotrypsin also failed to react with the monoclonal antibodies in either form. Eighteen octapeptides spanning the entire sequence of this region were synthesized on polyethylene pins, and epitopes of representative monoclonal antibodies were determined by ELISA. The reactivity of peptides suggest that a portion of the 25 mer peptide in AChE containing the active site serine is the primary epitope. It is not exposed on the surface of the enzyme and is most likely sequestered in a pocket-like conformation in the native enzyme.

Acetylcholinesterase↗
PubMed1989

Differences in structure and distribution of the molecular forms of acetylcholinesterase.

Two structurally distinct molecular forms of acetylcholinesterase are found in the electric organs of Torpedo californica. One form is dimensionally asymmetric and composed of heterologous subunits. The other form is hydrophobic and composed of homologous subunits. Sequence-specific antibodies were raised against a synthetic peptide corresponding to the COOH-terminal region (Lys560-Leu575) of the catalytic subunits of the asymmetric form of acetylcholinesterase. These antibodies reacted with the asymmetric form of acetylcholinesterase, but not with the hydrophobic form. These results confirm recent studies suggesting that the COOH-terminal domain of the asymmetric form differs from that of the hydrophobic form, and represent the first demonstration of antibodies selective for the catalytic subunits of the asymmetric form. In addition, the reactive epitope of a monoclonal antibody (4E7), previously shown to be selective for the hydrophobic form of acetylcholinesterase, has been identified as an N-linked complex carbohydrate, thus defining posttranslational differences between the two forms. These two form-selective antibodies, as well as panselective polyclonal and monoclonal antibodies, were used in light and electron microscopic immunolocalization studies to investigate the distribution of the two forms of acetylcholinesterase in the electric organ of Torpedo. Both forms were localized almost exclusively to the innervated surface of the electrocytes. However, they were differentially distributed along the innervated surface. Specific asymmetric-form immunoreactivity was restricted to areas of synaptic apposition and to the invaginations of the postsynaptic membrane that form the synaptic gutters. In contrast, immunoreactivity attributable to the hydrophobic form was selectively found along the non-synaptic surface of the nerve terminals and was not observed in the synaptic cleft or in the invaginations of the postsynaptic membrane. This differential distribution suggests that the two forms of acetylcholinesterase may play different roles in regulating the local concentration of acetylcholine in the synapse.

Acetylcholinesterase↗
PubMed1989

Molecular characterization of a neutralizing domain of the Japanese encephalitis virus structural glycoprotein.

Expression of antigenic fragments of the Japanese encephalitis virus envelope protein (E) in Escherichia coli has been used to define the boundaries of an antigenic domain that contains the binding sites for 10 anti-E monoclonal antibodies (MAbs). All of these antibodies neutralized the virus in vitro and some of them passively protected mice from a fatal virus challenge. We have shown previously that nine of these antibodies react with the antigenic determinants encoded by a 405 bp fragment of viral cDNA. To determine the amino acid sequences of specific determinants, truncated polypeptides were expressed as fusion proteins in E. coli following progressive Bal 31 exonuclease digestion of the 5' and 3' ends of the cDNA fragment. Examination of the immunoreactivity of these polypeptides revealed that the region from methionine 303 to tryptophan 396 was the shortest sequence capable of reacting with any of the 10 MAbs or with a polyclonal, antiviral hyperimmune mouse ascitic fluid. Biochemical tests showed that an intramolecular disulphide cross-linkage between cysteine 304 and cysteine 335 of the E protein sequence was required for presentation of the binding site(s) for these MAbs. Although this 95 amino acid antigenic domain appeared to be capable of forming several conformational neutralizing epitopes, it was not an effective immunogen for inducing neutralizing or protective antibodies in mice.

Amino Acid Sequence↗
PubMed1989

Monoclonal antibodies for dengue virus prM glycoprotein protect mice against lethal dengue infection.

Five murine monoclonal antibodies (Mabs) reactive against the prM glycoproteins of DEN-3 and -4 were used to passively protect mice in vivo against lethal challenge with homologous and heterologous dengue virus serotypes. Four of the 5 prM-reactive monoclonals cross-protected mice against heterologous challenge, whereas 1 protected against challenge with only the homologous serotype. Although in vitro binding to virions was readily demonstrated, only 2 of the prM Mabs had detectable neutralizing activity. The neutralizing activity could not be enhanced by anti-mouse immunoglobulin or complement. However, 4 of the 5 prM Mabs fixed complement. This is the first report of prM-specific Mabs that are protective in mice.

Antibodies, Monoclonal↗
PubMed1988

Immunochemical and structural similarities in toxin A and toxin B of Clostridium difficile shown by binding to monoclonal antibodies.

Clostridium difficile toxins A and B were shown to share immunochemical and structural features, including shared sequential epitopes. Nineteen hybridomas generated after immunization of mice with a mixture of toxoids produced monoclonal antibodies, all IgM(x), which bound to toxin A and toxin B in a solid-phase radioimmunoassay (RIA). None of the antibodies neutralized the cytotoxicity of either toxin, alone or in pairs, nor did they neutralize mouse lethality. The antibodies did not inhibit hemagglutination by toxin A, and none of those tested neutralized the toxin's enterotoxic activity. Studies of binding of antibodies to native toxins in the RIA showed that the antibodies differed in their recognition of the toxins. Many of the antibodies bound with higher avidity to toxin A than to toxin B. In Western blots, all the antibodies recognized both toxins in the native state; in addition, some antibodies recognized the minor cytotoxic species of toxin B. When the toxins were denatured and reduced, five antibodies bound to both toxins, five to A only, and nine to neither, demonstrating that the antibodies had different epitope specificities. Further structural comparisons were made by investigation of mol. wts, subunit structures and amino acid compositions. The native mol. wts of toxin A and toxin B, as determined by electrophoresis to equilibrium in 4-30% polyacrylamide gel electrophoresis (PAGE), were 430,000 and 368,000, respectively. Denatured and reduced toxins each had a single subunit of 315,000. Both toxins had about 50% hydrophobic amino acids.

Amino Acids↗
PubMed1988

Antibodies to cholesterol.

Cholesterol-dependent complement activation has been proposed as a factor that might influence the pathogenesis of atherosclerosis. Although antibodies to cholesterol conjugates have been reported, cholesterol is widely regarded as a poorly immunogenic substance. Monoclonal IgM complement-fixing antibodies to cholesterol were obtained in the present study after immunizing mice with liposomes containing high amounts of cholesterol (71 mol % relative to phosphatidylcholine) and lipid A as an adjuvant. Clones were selected for the ability of secreted antibodies to react with liposomes containing 71% cholesterol but not with liposomes containing 43% cholesterol. The antibodies also reacted with crystalline cholesterol in a solid-phase enzyme-linked immunosorbent assay. Binding of monoclonal antibodies to the surface of crystalline cholesterol was demonstrated by electron microscopy by utilizing a second antibody (anti-IgM) labeled with colloidal gold. The immunization period required to induce monoclonal antibodies was very short (3 days) and a high fraction of the hybrid cells (at least 70%) were secreting detectable antibodies to cholesterol. The results demonstrate that cholesterol can be a highly immunogenic molecule and that complement-fixing antibodies to cholesterol can be readily obtained.

Animals↗
PubMed1988

A monoclonal antibody for the specific diagnosis of plague.

A stable mouse-cell hybridoma was obtained that secretes an IgA monoclonal antibody reactive with the fraction 1 (F1) envelope antigen of Yersinia pestis. Titres of the antibody typically ranged from 1:32 768 to 1:65 536 in mouse ascitic fluids.The monoclonal antibody formed a line of precipitation when run against F1 antigen in Ouchterlony gel diffusion tests. In tests of 235 strains of Y. pestis, lines of identity occurred between the precipitates formed with a solution of purified F1 antigen and the F1 antigen produced by the plague strains. No precipitates formed for 65 strains that were incapable of elaborating F1 antigen. Specificity of the monoclonal antibody for strains of Y. pestis producing F1 was also indicated by negative results for 50 yersinia strains other than Y. pestis tested by an ELISA that used the antibody to capture antigen.Experiments to determine the shelf-life of the antibody were conducted over 3-4 years. When the monoclonal antibody was freeze-dried in vials, titre was retained for three years when the vials were stored at -70 degrees C but only for two months when they were stored at ambient temperatures. When the antibody was freeze-dried in wells of ELISA plates, sensitivity of the plates for capture of F1 antigen was preserved for four years when the plates were stored at -70 degrees C compared with two weeks for plates stored at room temperature. When a solution of the antibody was sealed in wells of ELISA plates and refrigerated at 4 degrees C, reactivity of the antibody and sensitivity of the plates were retained for a year.Alternatives for the application of this monoclonal antibody in ELISA and other plague diagnostic procedures are discussed.

Animals↗
PubMed1987

Detection of flavivirus antibodies in human serum by epitope-blocking immunoassay.

Human flavivirus group-reactive, dengue complex-reactive, and encephalitis virus complex-reactive antibodies were detected using epitope-blocking immunoassays in which the binding of selected mouse monoclonal antibodies to flavivirus antigens was blocked by human serum. When late (greater than 6 months after illness) convalescent sera were tested, the epitope-blocking immunoassays were superior to the hemagglutination inhibition test and comparable to the plaque reduction neutralization for identifying subjects immune to dengue, to Japanese encephalitis, or both viruses.

Antibodies, Viral↗
PubMed1986

Geographic classification of dengue-2 virus strains by antigen signature analysis.

Dengue-2 virus strains from different locations were compared by T1-RNAse-resistant oligonucleotide fingerprinting and antigen signature analysis. The latter technique involved construction of radioimmunoassays using monoclonal antibodies that recognize nine distinct dengue-2 type-specific and flavivirus cross-reactive epitopes over a range of antigen concentrations. A statistical method was used to align unknown dengue antigen concentrations in different strain preparations, allowing comparison of binding profiles. Twenty-six dengue-2 virus strains were separated into five distinct groups (topotypes) on the basis of unique RNA fingerprints. Two of these were represented by New Guinea C, the prototype virus isolated in 1944, and a Philippine strain; others were segregated on the basis of greater than or equal to 80% shared oligonucleotides into similarity groups representing Burma/Thailand (8 strains), Puerto Rico (12 strains), and Jamaica (4 strains). Signature analysis of the prototype and four geographic topotype strains revealed striking antigenic differences. In contrast, a high degree of antigenic similarity was found among strains from the same geographic region. Variation between antigenically distinct strains occurred at both type-specific and group-reactive epitopes, but the widest differences appeared at group-reactive determinants. Signature analysis provides a more rapid and simpler means than RNA fingerprinting of monitoring changes or new introductions of dengue virus populations in a geographic region.

Antibodies, Monoclonal↗
PubMed1986

Multisite monoclonal immunoassay for dengue viruses: detection of viraemic human sera and interference by heterologous antibody.

A monoclonal radioimmunoassay (RIA) was developed for detection of dengue virus in infected cell culture fluids and blood samples from dengue patients. Antibodies used to construct the RIA were selected on the basis of high binding avidity, the demonstration of synergism in competitive binding assays and empirical trials with different antibody combinations. Optimal binding of all four dengue virus serotypes was achieved by use of a flavivirus group-reactive and a dengue virus complex-reactive antibody as radiolabelled probe. A 'simultaneous sandwich' format and prolonged (18 h) incubation at 37 degrees C yielded optimal results. The limit of sensitivity of the RIA for detection of dengue type 2 virus was 2.7 log10 mosquito 50% infectious doses (MID50). The assay was tenfold more sensitive for dengue type 2 than for dengue types 1 and 3 viruses and 100-fold more sensitive than for dengue type 4 virus. Specificity, assessed using over 500 disease control human sera, was increased by addition of monoclonal anti-tetanus blocking antibodies, resulting in a false positive rate of only 0.2%. Heterologous dengue virus antibodies were shown to inhibit the RIA in assays performed with artificial immune complexes. Acute phase human sera containing 10(4.2) to 10(7.6) MID50 but no detectable antigen by RIA, were also shown to inhibit binding of the homologous dengue virus serotype; this effect was attributed to heterologous antibody from a prior infection. Among 116 viraemic sera from dengue patients, the RIA was positive in 43 to 47% of patients with dengue type 1, 2 or 3 infections but in only 10% of the dengue type 4 cases. Virus was more frequently detected in cases of primary infection (54%) than in cases of superinfection (16%). Despite the limitations imposed by immunological interference, the antigen capture RIA appears useful as a rapid diagnostic technique for dengue surveillance.

Aedes↗
PubMed1986

Molecular mimicry: frequency of reactivity of monoclonal antiviral antibodies with normal tissues.

More than 600 monoclonal antiviral antibodies made against 11 different viruses were screened against 14 different organs from normal uninfected mice. Of these antiviral antibodies, 21, or approximately 3.5%, reacted with specific cells in these organs. Several of these antibodies were of the multiple-organ-reactive type and recognized antigens in more than one organ. It was concluded that the reactivity of monoclonal antiviral antibodies with normal tissues is a common phenomenon.

Animals↗
PubMed1984

Use of an enzyme-linked immunosorbent assay to measure antigenaemia during acute plague.

An enzyme-linked immunosorbent assay (ELISA) was developed to measure concentrations of the specific F1 antigen of the plague bacillus in biological fluids. The assay employed a monoclonal antibody to capture the antigen. Sensitivity of the assay was 0.4 ng of F1 antigen. ELISA-inhibition was used to confirm the specificity of the reactions.This assay detected F1 antigen in two of ten sera from patients with acute bubonic plague and indicated that antigenaemia in man during plague may reach levels of 4-8 mug of F1 antigen per ml of serum.The probability for a correct serodiagnosis of plague was improved when the patients' sera were tested for both antibody and antigen. Two patients with antigenaemia did not have antibody, while two patients with antibody lacked antigenaemia.

Antigens, Bacterial↗
PubMed1984

Heterogeneity of infection enhancement of dengue 2 strains by monoclonal antibodies.

Seven dengue (DEN) 2 virus strains were studied for antibody-dependent enhancement (ADE) of infection in P388D1 mouse macrophage-like cells by using a panel of five DEN 2 monoclonal antibodies. DEN 2 strains were of diverse temporal, geographic, and disease origins. By hemagglutination inhibition and a plaque-reduction neutralization test in LLC-MK2 cells, two of the monoclonal antibodies were type specific and three were flavivirus group reactive. In LLC-MK2 cells, the seven DEN 2 viruses each were neutralized by all five monoclonal antibodies. In P388D1 cells, two DEN 2 strains were enhanced by only three monoclonal antibodies, two by four antibodies, and three by all five antibodies, demonstrating that in some instances enhancement is epitope related and not a concentration-dependent function of virus-antibody interactions. However, ADE did not segregate with determinants exhibiting either the flavivirus group or the dengue type specificity. The presence or absence of enhancement determinants on DEN 2 strains did not correlate with the geographic origin of virus or the severity of disease yielding the strain. The heterogeneous distribution of enhancement determinants may provide a valence mechanism contributing to a multiple increase of infection enhancement in macrophages.

Animals↗
PubMed1983

Antigenic and structural differences in the catalytic subunits of the molecular forms of acetylcholinesterase.

A mixture of the 5.6S hydrophobic dimer and the asymmetric, tail-containing (17 + 13)S forms of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) from Torpedo californica was used to immunize mice, and spleen cells from these mice were used to produce nine hybridoma lines secreting antibodies against acetylcholinesterase. Antibodies from one of the lines showed a 100-fold greater affinity for the 5.6S species when compared with the catalytic subunits of the (17 + 13)S species. This difference in specificity was retained after denaturation of the two acetylcholinesterase species. Another line produced antibody directed only to structural subunits of the (17 + 13)S species, whereas the remaining seven antibodies exhibited nearly equivalent crossreactivity for all of the forms of acetylcholinesterase. Tryptic peptides were generated from the catalytic subunits of the 5.6S and tail-containing acetylcholinesterase species, and high-pressure liquid chromatographic profiles show at least two distinct peptides in the catalytic subunits for each enzyme species. Some of these peptides exhibit retention times different from those of the identified glycopeptides. Thus, it is likely that the catalytic subunits of two molecular forms of acetylcholinesterase differ in primary structure and sites of antigenicity.

Acetylcholinesterase↗
PubMed1983

Isolation and characterization of monoclonal antibodies to Shiga toxin.

Hybridoma cell lines which produce monoclonal antibodies to Shiga toxin from Shigella dysenteriae 1 were prepared. The monoclonal antibodies were all of the immunoglobulin G1 isotype and differed in their ability to neutralize cytotoxicity and to bind to Shiga toxin in a solid-phase radioimmunoassay. When used for immunoblot analysis, these antibodies were able to identify specifically both nicked and unnicked Shiga toxin in crude lysates of S. dysenteriae.

Animals↗
PubMed1983

Rapid identification of dengue virus isolates by using monoclonal antibodies in an indirect immunofluorescence assay.

Type-specific monoclonal antibodies prepared against the four dengue (DEN) virus serotypes were evaluated for their ability to identify low-passage human and mosquito isolates from Jamaica and West Africa by an indirect immunofluorescence assay. Serotyped human isolates from Jamaican dengue fever patients included 12 DEN-1, two DEN-2, and five DEN-4 viruses. Viruses from West Africa included 84 DEN-2 mosquito strains as well as two DEN-1 and one DEN-2 from humans. Results obtained using the immunofluorescence assay were consistent with virus identifications obtained using the more classical but costly and time-consuming plaque-reduction neutralization test. More viral isolates and higher virus yields were obtained using the C6/36 clone of Aedes albopictus cells rather than LLC-MK2 (monkey kidney) cells. Dengue type-specific monoclonal antibodies detected prototype viral antigens 24-48 hours postinfection in C6/36 cells. This is the first time that monoclonal antibodies have been used to serotype low-passage flavivirus isolates.

Aedes↗