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J M Coll

Publications and source records attributed to J M Coll.

At least 55 records · Page 3Linked to original sources
PubMed1993

Characterization of a soluble hemagglutinin induced in African swine fever virus-infected cells.

Hemadsorption (Had) of erythrocytes to the surface of African swine fever virus (ASFV)-infected cells is a well-known phenomenon but hemagglutination of pig erythrocytes in the supernatant of ASFV-infected cells has not been reported before. We report here the discovery of a pig erythrocyte-agglutinating activity released to the in vitro cell culture medium by cells infected with some isolates of ASFV. This finding allowed the identification and characterization of a soluble hemagglutinin (HA) molecule that could be separated from the ASFV particles either by ultracentrifugation or by gel-permeation chromatography. The HA was inactivated by agents known to affect protein conformation such as heat, beta-mercaptoethanol, urea, and guanidine isothiocyanate. Glycosylation seemed to be of importance since treatment of HA with glycosidase F inhibited the hemagglutinating activity and HA could be partially purified by affinity chromatography on immobilized concanavalin A. When native it had an estimated molecular weight of 300 kDa by gel-permeation chromatography yielding 51-kDa protein monomers under denaturing conditions as identified by immunoblotting. Preliminary attempts to correlate the induced anti-HA serum antibodies with viremia or infection-inhibition serum antibodies after infection of pigs with attenuated ASFV or immunization with purified HA are also reported.

African Swine Fever↗
PubMed1993

The in vitro infection of the hematopoietic stroma of trout kidney by hemorrhagic septicemia rhabdovirus.

Viral hemorrhagic septicaemia virus (VHSV) infected the hematopoietic stromal cells (7,8) derived from pronephritic tissue of the rainbow trout, Oncorhynchuss mykiss, W., at their ninth passage in vitro. Viral infection resulted in the development of lytic cytopathic effects on confluent in vitro tridimensional network stromal cell cultures. Replication of VHSV in the stromal cell cultures was demonstrated by the increase in infectivity by epithelioma papulosum cyprini (EPC) cell culture assays and by the increase of the nucleoprotein antigen of VHSV by ELISA. By using anti-VHSV monoclonal antibodies (MAbs), flow cytometry studies demonstrated that only the infected stromal cells contained cytoplasmic viral antigens. The lytic infection of trout hematopoietic stromal cells in vitro could be relevant to the hemorrhagic pathology seen in the kidney of fish infected with VHSV.

Animals↗
PubMed1993

Enhancement of fish mortality by rhabdovirus infection after immunization with a viral nucleoprotein peptide.

A similar sequence to a mouse immunodominant CTL peptide (SYVLQGN, single-letter amino acid code, conserved amino acids underlined) identified in the nucleoproteins of several strains of vesicular stomatitis virus (VSV) (37) was found in the nucleoproteins of viral hemorrhagic septicemia virus (VHSV) of salmonid fish (GYVYQGL in VHSV 07.71 and GYVYQGS in VHSV Makah) and not in the nucleoproteins of other rhabdoviruses. The in vivo immunization of fingerling salmonid fish (rainbow trout Onchorynchus mykiss, W) with this VHSV peptide and their subsequent challenge with VHSV resulted in the enhancement rather than in the reduction of fingerling trout mortality. Possible implications for the development of subunit vaccines against VHSV are discussed.

Amino Acid Sequence↗
PubMed1992

Neutralizing-enhancing monoclonal antibody recognizes the denatured glycoprotein of viral haemorrhagic septicaemia virus.

Two monoclonal antibodies (MAbs), 1H10 and 1F10, have been selected against the denatured glycoprotein of the viral haemorrhagic septicaemia virus (VHSV) of salmonids by immunoblotting. Three reference VHSV serotypes (F1, F2, 23.75) and five VHSV isolates from either different host species (trout, salmon, barbel) or geographical locations in Spain reacted with both MAbs by ELISA. In vitro neutralization of all VHSV serotypes but not infectious haematopoietic necrosis virus (IHNV) was obtained only with MAb 1H10. However, when the MAb 1H10 (but not the non-neutralizing Mab 1F10) concentration was below 100 micrograms/ml rather than neutralizing VHSV infectivity, plaque counts increased 2-3-fold. MAb 1H10 is unique in that it has both, an in vitro enhancing infectivity effect (not described yet for VHSV) and it recognizes denatured G protein in contrast to other previously described neutralizing MAbs against VHSV.

Animals↗
PubMed1992

In vitro studies and in vivo immunisation with the first viral haemorrhagic septicaemia viruses isolated in Spain compared to international reference serotypes.

The first five viral haemorrhagic septicaemia virus (VHSV) isolates found in Spain were examined for in vitro growth characteristics, neutralisation by trout antiserum and immunisation challenge of trout fingerlings by water immersion. The viruses had come from different host species in various geographical locations in different years. Three reference VHSV serotypes (F1, F2 and 23.75) were also included in the study. There appeared to be little relationship between the in vivo delay of mortality or the protection of the immunised trout after challenge and the in vitro characteristics studied. In contrast to the in vitro results, the in vivo delay of mortality suggested a closer relationship of the Spanish VHSV isolates to the F2 serotype than to the F1 or the 23.75 serotypes. If the final protection figures are analysed, however, there could be three or four groups of viruses.

Animals↗
PubMed1992

Mitogen-induced proliferation of trout kidney leucocytes by one-step culture in fibrin clots.

This work describes a trout kidney leucocyte mitogen-stimulation assay using fibrinogen/thrombin. Cloning was obtained by only one step instead of by two steps, as required by the agar method described to date. Cultures were stimulated not only with phytohemagglutinin (PHA) and Escherichia coli lipopolysaccharides (LPS) but also with Concanavalin A (Con A) and six LPS from aquatic pathogenic bacteria. The number of colony-forming cells detected, and their morphological type depended on the mitogen, the time of incubation and the trout. PHA was the best inducer of trout kidney leucocyte colony formation followed by Con A, giving rise to four different homogeneous types of colonies formed by large-nucleated cells, cells with eccentric nuclei, multinucleated cells and lymphocytes.

Animals↗
PubMed1992

Susceptibility of trout kidney macrophages to viral hemorrhagic septicemia virus.

Viral hemorrhagic septicemia virus (VHSV) lysed the macrophages from rainbow trout kidney cultures either isolated by plastic adherence or stimulated with purified glycoprotein G from VHSV. The trout macrophages supported the replication of VHSV as tested by cell culture and by sandwich ELISA of the supernatants from infected cultures. VHSV-infected macrophages showed a decrease in both acridine-orange fluorescence and average size. Immunofluorescence studies with flow cytometry showed positive membrane staining with monoclonal antibodies (MAbs) anti-N and anti-G VHSV. These findings open the possibility of using trout macrophages as presenting cells to study the possible existence of helper or cytotoxic epitopes relevant to the protection of trout against VHSV.

Animals↗
PubMed1992

Detection of hemorrhagic septicemia virus of salmonid fishes by use of an enzyme-linked immunosorbent assay containing high sodium chloride concentration and two noncompetitive monoclonal antibodies against early viral nucleoproteins.

Inclusion of high-ionic strength buffers helped us to develop a sandwich ELISA to detect hemorrhagic septicemia virus (HSV) in cell culture and infected trout tissue extracts. For maximal sensitivity of 0.1 to 0.2 ng/well/100 microliters or about 10 to 50 TCID50/well/100 microliters, trout extracts were diluted 1:1 and assayed for the earliest synthesized nucleoprotein N. Simultaneous binding of the N protein from HSV in the sample to the wells coated with monoclonal antibody (2D5 against the N protein) and to the peroxidase-labeled monoclonal antibody (2C9 against the N protein) proceeded during a 2-hour incubation at 20 to 22 C (room temperature). The response was linear between 6 to 60 ng/well of purified virus. Monoclonal antibodies used were noncompetitive with each other and reacted with F1, F2, 23.75, and 5 Spanish isolates of HSV, but not with infectious hematopoietic necrosis or infectious pancreatic necrosis viruses. Tissue specimens with low content of HSV virus may now be assayed directly without use of cell culture, rapidly, and with high precision, during the acute phase of the disease in salmonid fishes.

Animals↗
PubMed1991

The free nucleocapsids of the viral haemorrhagic septicaemia virus contain two antigenically related nucleoproteins.

A protein of 34 kDa (Nx) was induced in vitro by the infection of fish cell cultures with the rhabdovirus agent of viral haemorrhagic septicaemia (VHS) of the trout. This protein only appeared as a major component in concentrated or intracellular labeled VHS virus but not in purified VHS or in the related infectious haematopoietic necrosis virus. That Nx protein is antigenically related to the nucleoprotein of purified virus was shown by its reaction with four anti-nucleoprotein monoclonal antibodies (at least 3 of them reacting non competitively against different epitopes) and by immunoprecipitation with polyvalent international reference sera. The Nx protein was shown to be specifically associated with free non-infective particles isolated by ultracentrifugation which were confirmed to be nucleocapsids by electron microscopy.

Animals↗
PubMed1991

Stimulation of adherent cells by addition of purified proteins of viral hemorrhagic septicemia virus to trout kidney cell cultures.

Purified proteins of the virus causing viral hemorrhagic septicemia in the trout were added to cultures on semisolid medium of leukocytes obtained from either healthy or immunized rainbow trout. Adherent cells were specifically stimulated by the glycoprotein of the viral spikes and, to a lesser extent, by the nucleoproteins. In contrast, a specific memory response was associated more with the nucleoproteins than with the glycoprotein when leukocytes from trout immunized with the virus were employed. These results suggest the necessity of employing both proteins in subunit vaccination trials and the possibility of using this assay to select the proper epitopes for genetically engineered proteins during subunit vaccine development.

Animals↗
PubMed1989

Addition of reducing agents to the peroxidase-o-phenylenediamine buffer reduces background of enzyme immunoassays.

The concentrations of o-phenylenediamine (OPD), H2O2, citrate and H+ in a substrate buffer for peroxidase immunoassays were optimized for minimal background. The background was reduced 2-3 fold with 5.5 mM OPD, 3 mM H2O2, 150 mM citric acid/sodium citrate, pH 4.8, and the reproducibility interassay was increased. A further 3-5 fold reduction of the background was obtained by the addition of 1.5 mM acetanilide, 0.14 mM beta-mercaptoethanol and 5 mM nitrilotriacetic acid to the substrate buffer. This low-background substrate buffer allows increased sensitivity and lowers the interassay variation coefficient. It has been used successfully in peroxidase immunoassays of human C-reactive protein, human antiestreptolysin and human rheumatoid factor.

Acetanilides↗
PubMed1989

Isolation of lipid-free C-reactive protein by affinity chromatography.

Human C-reactive protein purification has been hampered by its association with lipids. Isolation of pure lipid-free C-reactive protein was obtained by a three step procedure. First, partially lipid-free C-reactive protein was obtained by affinity chromatography from ascitic fluids; second, lipid-bound proteins were eliminated by calcium-dependent precipitation; and third, lipid-free pure C-reactive protein was obtained by affinity re-chromatography of the supernatant. A 46-50% yield of lipid-free C-reactive protein was obtained compared with the 14.7% obtained by the old method of extraction with lipid solvents.

C-Reactive Protein↗
PubMed1988

Cloning of myelomas and hybridomas in fibrin clots.

Myelomas and hybridomas were observed to proliferate normally when trapped in a fibrin clot. The fibrin clot was obtained by including fibrinogen in the cell culture medium and thrombin in the plastic dish. A clot formed within seconds and cloning efficiency was around 100%. This technique has all the advantages of semi-solid medium cloning but avoids toxicity to the cells and the exposure to high temperature associated with the soft-agar cloning technique.

Animals↗
PubMed1988

Selection and performance of monoclonal anti-C-reactive protein in ELISA quantitative assay.

Nine monoclonal anti C-reactive protein (CRP) antibodies were tested as peroxidase-conjugates in a CRP binding to a solid-phase phosphorylethanolamine (PE) assay. One monoclonal antibody was selected due to its high yield of conjugate, titre and stability. The use of monoclonal antibodies increased assay sensitivity, precision and allowed assay simplification by the simultaneous rather than sequential incubation of CRP and conjugate. Comparison with nephelometry and CRP binding to PE assay using polyclonals gave correlations greater than or equal to 0.9. By using monoclonal conjugates to assay human sera, healthy adult CRP levels were found to be lower than by using polyclonal conjugates. Samples with very low CRP contents might now be assayed with higher precision.

Adult↗
PubMed1988

Immunochemical recognition of the binding of C-reactive protein to solid-phase phosphorylethanolamine.

The influence of polyclonal and monoclonal antibodies, trypsin digestion and mercaptoethanol treatment of C-reactive protein (CRP) in the CRP binding to solid-phase phosphorylethanolamine (PE) has been investigated. Nine monoclonal antibodies reacting with CRP could be divided into at least 2 well-defined groups: one group of 6-7 monoclonals interfering with the binding of CRP to PE (mainly represented by monoclonal 2) and the not interfering with the binding of CRP to PE (mainly represented by monoclonal 5). Trypsin digestion resulted in sequence identified CRP fragments still able to bind to PE and detectable by monoclonal 5 but not by monoclonal 2. On the other hand, binding of CRP to PE was abolished by mercaptoethanol treatment. These results, together with the estimation of the extent of the antigenicity of the PE binding site and the characteristics of the hydrophobicity profile of CRP, suggest that most of the hydrophilic sequences contribute to the PE binding region except a non-overlapping region defined by monoclonal 5. Most probably, some of these sequences are located inside or around the internal bisulphide bridge of each monomer of the pentameric CRP.

Antibodies, Monoclonal↗
PubMed1988

Qualitative screening of C-reactive protein by latex in microtiter trays.

A microtiter-latex anti-C reactive protein method is described for screening large number of samples. The high percentage of false positives found with the slide-latex anti-C reactive protein method was reduced about 6 fold by the use of the described method. The use of microtiter trays, dilution of serum in one step and decreasing time of assay make this method simple, specific, rapid and easy to perform without sophisticated equipment.

C-Reactive Protein↗
PubMed1988

Development of a fast solid-phase enzyme immunoassay for C-reactive protein.

A fast sandwich enzyme immunoassay has been developed for C-reactive protein (CRP). This method can be used for screening CRP concentration in large numbers of samples providing a non precipitation, non agglutination and non radioactive alternative for assessment of human CRP. Advantages over previously reported CRP sandwich assays include: assay time was reduced from 4 1/2 h to 45 min, incubations were made at room temperature instead of 37 degrees C and serum dilutions required were 100-400 fold instead of 10000-20000 fold. Correlations were good with both nephelometry and phosphorylethanolamine binding assay. The 45% false positives found with the slide-latex anti C-reactive protein method were reduced to 0% by the use of the described method.

C-Reactive Protein↗