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K C McCullough

Publications and source records attributed to K C McCullough.

69 records · Page 4Linked to original sources
PubMed1985

Alteration in antibody reactivity with foot-and-mouth disease virus (FMDV) 146S antigen before and after binding to a solid phase or complexing with specific antibody.

This paper describes the reactions of a number of monoclonal antibodies produced against purified whole virions of foot-and-mouth disease virus in 3 different enzyme immunoassay systems. The first system used whole virus bound non-covalently to microplates; the second used whole virus trapped by a polyclonal antibody which was bound to microplates; and the third allowed the monoclonal antibodies to react with the whole virions in suspension (liquid phase) before trapping by the solid-phase-bound polyclonal antibody. Different reactions with panels of monoclonal antibodies were observed depending on which system was used. Such variations in reactivity give an insight into the alterations in the expression of virus epitopes in the different enzyme immunoassay systems. The reactions of selected monoclonal antibodies were used to illustrate these changes and the results compared to those obtained in similar systems using polyclonal antisera produced against isolated virion polypeptides.

Animals↗
PubMed1985

A liquid-phase ELISA and its use in the identification of epitopes on foot-and-mouth disease virus antigens.

An enzyme-linked immunosorbent assay was developed which would detect antigen/antibody reactions in liquid-phase; that is under test conditions which should not alter the structure or reactivity of either antigen or antibody. This liquid-phase ELISA was at least as efficient, both qualitatively and quantitatively, as the double sandwich or antigen-trapping ELISA (Crowther and Abu Elzein, 1979), but six to eight times more sensitive than the indirect ELISA. The liquid-phase test can be used to assay both antisera and hybridoma cultures, and will show the greatest discrimination of antibody specificities. Since the liquid-phase ELISA will detect antibody/antigen reactions which are of direct relevance to the protective immune response in vivo (opsonisation; neutralisation of pathogen infectivity), it is recommended that this assay should be used to characterise the antibody reactivity of antisera and hybridoma culture supernatants.

Animals↗
PubMed1985

Anti-idiotope vaccines: can they exist?

Idiotopes and idiotypes are determinants on antibody molecules and lymphocyte receptors which confer a degree of uniqueness to these molecules; a number of the idiotypes are associated with the paratope (antigen-combining site) of the antibody. Since idotypic determinants can be recognized as 'foreign' by the autologous immune system, both antibodies and lymphocyte receptors which are anti-idiotypic in function can be found. Hence, anti-idiotype antibodies against paratope-associated idiotypes (Idp) would themselves have paratopes of similar conformation to the epitope on the antigen with which the Idp-bearing antibody reacted. These are the 'internal image' anti-idiotype antibodies. Vaccination of animals with such anti-idiotype preparations should result in the production of Idp-bearing antibody molecules. If these Idp antibodies were protective against infection by a particular pathogen, then the anti-idiotope [anti-(Idp)] preparation would be considered a surrogate antigen. Such anti-idiotype surrogate antigens have been identified, but they were polyclonal and their composition is not, as yet, completely understood. Monoclonal 'internal image' anti-idiotype antibodies have not proved to be consistently successful surrogate antigens. This may reflect a requirement for a minimum avidity of reaction, and antibody specificities in addition to that of the 'internal image'. Further insight into the composition of the polyclonal surrogate antigen is required before elaboration on an 'anti-idiotope vaccine' can be provided.

Animals↗
PubMed1985

Is rinderpest virus the archevirus of the Morbillivirus genus?

Groups of 6-39 monoclonal antibodies identifying 3-18 distinct epitopes on the nucleoprotein (NP), polymerase (P), hemagglutinin (H; equivalent in canine distemper and rinderpest viruses), and fusion (F) components of measles and canine distemper viruses were characterized in immunofluorescence tests with fixed Vero cell cultures infected with measles, canine distemper and rinderpest viruses. The majority of NP-specific monoclonal antibodies reacted with all three viruses, but one-third of the antibodies only reacted with the homologous virus. A few antibodies detected epitopes uniquely shared between either measles and rinderpest viruses or canine distemper and rinderpest viruses. Of the P-specific antibodies, two-thirds only reacted with the homologous virus, one antibody detected an epitope shared between canine distemper and rinderpest viruses, and the rest reacted with all three viruses. Also, the majority of antibodies against the H component were type-specific, but four antibodies reacted both with measles and rinderpest viruses. In contrast, the F component was antigenically highly conserved. 17 of 21 antibodies against this component reacted with all three viruses; one antibody reacted only with measles and rinderpest virus F components, and three antibodies reacted only with the homologous virus. No monoclonal antibody of any specificity selectively reacted with only measles and canine distemper viruses. Furthermore, the measles virus H component appeared to be more closely related to the equivalent rinderpest virus component than to the canine distemper virus component. Thus, it is proposed that rinderpest virus is the archevirus of the morbillivirus group from which canine distemper virus was first to evolve and, more recently (perhaps about 5,000 years ago), measles virus.

Antibodies, Monoclonal↗
PubMed1984

The standardization of a 'spot-test' ELISA for the rapid screening of sera and hybridoma cell products I. The determination of the optimum buffering system.

Using the different commercially available enzyme-linked immunosorbent assay (ELISA) plates, several sources of albumin were tested along with Tween 20 as supplements to the diluting buffer in ELISA for their ability to minimize non-specific reactions. There was an obligate requirement for Tween 20 (0.05%) but the different albumin sources had varied effectiveness on each of the different ELISA plates. In general, however, the optimum buffering system was concluded to be phosphate-buffered saline supplemented with 0.05% (v/v) Tween 20 and 3% (w/v) lactalbumin yeast hydrolysate or bovine serum (plasma) albumin fraction V.

Albumins↗
PubMed1984

The standardization of a 'spot-test' ELISA for the rapid screening of sera and hybridoma cell products II. The determination of binding capacity, binding ratio and coefficient of variation of different ELISA plates in sandwich and indirect ELISA.

The different commercially available enzyme-linked immunosorbent assay (ELISA) plates were compared for their binding capacity for purified foot-and-mouth disease virus antigen or IgG, their binding ratio (a measure of the efficiency with which positive and negative serum samples may be distinguished), and their coefficients of variation within a plate, between plates and between batches of plates. No one plate could be described as having ideal characteristics, and the choice of ELISA plate depends on the use to which the ELISA is being put. For our purposes, viz. a 'spot-test' which rapidly and efficiently detects specific antibody when the levels of that antibody are low (hybridoma culture supernatants) or when the antibody is contaminated with other 'interfering' proteins (high concentrations of serum), we found that most of the PVC plates and, of the polystyrene plates, the Nunc Immunoplate I and Dynatech M129B plates performed well. The lowest coefficients of variation were obtained using Nunc Immunoplate I, Dynatech M129B and Falcon 3912 plates.

Antibodies↗
PubMed1984

Induction of antibody to foot-and-mouth disease virus in presensitized mouse spleen cell cultures.

Cultures of spleen cells from immunized mice were stimulated in vitro by soluble preparations of purified foot-and-mouth disease virus. Virus-specific antibody, as detected by an enzyme-linked immunosorbent assay, was produced by immune spleen cells but not by normal, nonimmune cells. The optimal specific response was obtained with 1 microgram of virus per ml of culture; as the virus concentration was increased, the production of specific antibody was reduced. For very low concentrations of virus (less than 0.01 microgram per culture), there was tentative evidence of suppression of the specific antibody response. The levels of specific antibody induced were dependent on the source and number of plastic-adherent cells present in the cultures. We intend to use this model system to study further the basis of immunity to foot-and-mouth disease virus.

Animals↗
PubMed1983

Hybridoma cell lines secreting monoclonal antibodies against foot-and-mouth disease virus. 1. Cell culturing requirements.

The production of hybridoma cell lines secreting antibody against foot-and-mouth disease virus (FMDV) was more difficult than the production of similar cell lines secreting antibody against vesicular stomatitis virus or measles virus. A rapid and efficient protocol for the selection and culturing of 'anti-FMDV' hybridoma cultures was therefore developed and is described. This required the determination of the optimal culture medium (commercially available), source of serum supplement, line of myeloma cells, type of culture and routine for the subculturing of the hybridoma cells. The protocol consisted of fusion between immune splenocytes and NS-1 mouse myeloma cells, seeding into the wells of 24-well (24W) plates, culturing in RMPI 1640 medium supplemented with either foetal or donor calf serum, and passaging through 24W plates, 6W plates and 100 ml flasks (20 ml medium), respectively. The time at which aminopterin was added to kill unfused myeloma cells was also critical, with the optimum time being 24 h after fusion. In contrast, the B lymphocyte growth stimulant (2-mercaptoethanol) had no beneficial effects on the growth of the hybridomas.

Animals↗
PubMed1983

Hybridoma cell lines secreting monoclonal antibodies against foot-and-mouth disease virus (FMDV). II. Cloning conditions.

Three methods of cloning hybridoma cells--picking colonies from the masterplate, limit dilution cloning, and cloning in semi-solid medium over macrophage (m phi) feeder layers--were compared. Cloning in semi-solid medium was found to be the most efficient and reliable, especially with our relatively slow growing anti-foot-and-mouth disease virus (FMDV) antibody secreting hybridoma cells. The optimum culture dish for this cloning was the 6-well (6W) dish (well diameter 1.5 cm), while the optimum cloning procedure was 10 to 10(2) hybridoma cells in 1% (w/v) methylcellulose in Dulbecco's minimal essential medium (DMEM) supplemented with 50% (v/v) conditioned medium, 10% (v/v) foetal or donor calf serum and 2 mM glutamine, over a 24-48 h old culture of syngeneic m phi in each well of the 6W plate. This could, however, produce problems in that colony formation was sometimes 'loose', and confident picking of individual colonies was impaired. Such a problem could be avoided by using a solid agar interface of 1% (w/v) agar Noble in DMEM or RPMI 1640 medium between the feeder cells and the hybridoma cells in semi-solid medium.

Animals↗
PubMed1983

Characterization of a non-syncytiogenic autonomously replicating variant of measles virus.

A variant, derived from both measles and subacute sclerosing panencephalitis virus preparations, incapable of producing intercellular fusion or syncytia typical of measles virus infections is described. This non-syncytiogenic (N-S) virus was easily detected using immunofluorescence, was genetically stable and replicated autonomously. The variant was defective in the production of both infectious virus and haemagglutinin in Vero and Hep2 cells. Persistent infections in Hep2 cells were readily established without the cooperation of defective interfering particles or temperature-sensitive mutants, although interferon could be detected. It is possible that such N-S variants may be involved with the regulation of persistent infections by measles virus.

Animals↗
PubMed1982

Monoclonal antibodies against foot-and-mouth disease virus 146S and 12S particles.

This paper is the first description of monoclonal antibodies specific for foot-and-mouth disease virus (FMDV). Using these antibodies, it was possible to distinguish similar and unique antigenic sites on complete (146S) or subunit (12S) virus particles. Some of these monoclonal antibodies could also distinguish between 12S subunits made by acid degradation of 146S virus (12SA) and those viral components found in infected cell lysates which sediment in sucrose density gradients as "12S subunits" (12SN).

Animals↗
PubMed1979

Dermatitis.

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Dentists↗
PubMedDate not supplied

Analyses of the primary in vitro responsiveness of non-immune porcine peripheral blood mononuclear cells with reference to immunization by African swine fever virus antigen and treatment with leucine methyl ester.

Peripheral blood mononuclear cells (PBMC) from non-immune pigs were immunized in vitro using African swine fever (ASF) virus antigen with concomitant mitogenic stimulations known to have varying effects on B and T lymphocyte activity. None of these conditions, including those previously reported as being successful for the in vitro immunization of non-immune porcine PBMC with ASF virus antigen, supported the induction of specific antibody. Due to the reports on in vitro immunization of human PBMC, attempts were made to control suppressor cell activity in the porcine PBMC from non-immune pigs through L-leucine methyl ester (Leu-OMe) treatment. Upon immunization of the Leu-OMe treated PBMC with ASF virus antigen, concomitant with mitogen or cytokine stimulations, no specific antibody production was detected. Nevertheless, aspecific porcine immunoglobulin secretion was observed. Further analysis of the PBMC responsiveness demonstrated that 2.5 mM Leu-OMe (the dose recommended for use with human PBMC) suppressed mitogen-induced porcine lymphocyte proliferation, but in the absence of any detectable cytotoxicity. In fact, both anti-ASF virus antigen specific immunization and stimulation with "T-lymphocyte" mitogens were suppressed, whereas pokeweed mitogen stimulation of B lymphocyte aspecific immunoglobulin secretion was unaffected. Consequently, it was not possible to immunize in vitro non-immune porcine PBMC with ASF virus antigen as had been previously reported, nor was it possible to transfer the technology successfully used with non-immune human PBMC to the in vitro stimulation/immunization of non-immune porcine PBMC. The furtherance of this work will require greater insight into the immunobiological parameters and dynamics of the stimulation of non-immune porcine peripheral blood leukocytes, which are not as simple as previously reported, nor the same as identified with human PBMC.

African Swine Fever↗