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Biomedical subjects

J R Crowther

Publications and source records attributed to J R Crowther.

At least 37 records · Page 2Linked to original sources
PubMed1991

Evaluation of a trapping ELISA for the differentiation of foot-and-mouth disease virus strains using monoclonal antibodies.

A trapping enzyme-linked immunosorbent assay (ELISA) has been evaluated for the differentiation of foot-and-mouth disease virus (FMDV) strains using a panel of seven anti-serotype O monoclonal antibodies (MAbs). The variation of results within and between tests performed on the same day and on different days was examined using three strains of FMDV. Criteria for establishing antigenic differences between the strains as defined by the individual MAbs are proposed based on the variability measured, which can be used as standards by workers performing this test with other MAbs and FMDV strains.

Animals↗
PubMed1990

Characterisation of the European seal morbillivirus.

The ELISA test originally developed for the detection of serum antibodies to rinderpest virus has been shown to detect cross-reacting antibodies in sera of diseased common and grey seals. Analysis of sera collected from various seal populations is in progress to establish the correlation between different morbillivirus neutralisation tests, ELISA tests and the disease status of the animals. RNA purified from post-mortem tissues removed from diseased seals has been analysed by hybridisation with cloned cDNAs made to various genes of canine distemper, peste des petits ruminants, rinderpest and measles viruses. This study has confirmed the presence of RNA sequences characteristic of a morbillivirus, but shown that the virus is not identical with any known morbillivirus. Work is in progress to determine the nucleotide sequences of clones carrying inserts homologous to morbillivirus genes isolated from cDNA made on a template of infected seal tissue RNA.

Animals↗
PubMed1990

Evaluation of the use of chromogenic and fluorogenic substrates in solid-phase enzyme linked immunosorbent assays (ELISA).

Fluorogenic and chromogenic substrates were used in direct and trapping enzyme-linked immunosorbent assays (ELISA) for the detection of mouse IgG and foot-and-mouth disease virus (FMDV). The detection limits for both antigens were compared using different combinations of enzymes and substrates. Various times and concentrations of chemicals were used to obtain maximum sensitivity for both systems. Similar sensitivities were found using fluorogenic and chromogenic substrates. Tetramethyl benzidine substrate for horse-radish peroxidase enzyme conjugates was found to attain the highest sensitivity levels for chromogenic assays (0.12 ng IgG/ml and 1.0 ng/ml FMDV respectively), after 10 min incubation. Of the two fluorogenic enzyme/substrates studied, B-galactosidase was the most sensitive but required extended incubation times (2-3 h) as compared with chromogenic systems. Special microplates for fluoro-immunoassay (FIA) were compared with conventional microplates and no advantage was found to justify their use. An alkaline phosphatase anti-guinea-pig conjugate was used to confirm the equivalence of fluorogenic and chromogenic substrates in terms of sensitivity. A comparison of the amount of signal generated using various concentrations of enzyme in the absence of antigen was made for two different alkaline phosphatase conjugates to obtain theoretical sensitivity limits. One possible advantage of fluorogenic substrates is that high binding ratio can improve the confidence in discrimination of positive results.

Animals↗
PubMed1990

A competitive ELISA for the detection of group-specific antibodies to African horse sickness virus.

A competition enzyme-linked immunosorbent assay (ELISA) has been developed for the rapid identification and quantification of antibodies against African horse sickness (AHS) in sera from solipeds. The data showed the ELISA to be sensitive, specific and reliable. More than 1600 sera from 37 different countries were examined and results compared with those obtained by agar gel immuno-diffusion (AGID) tests. In no case did any of 775 sera from countries where AHS has never been reported and where AHS vaccines are not used, record an ELISA titre greater than 4. A titre equal to or greater than 8 was considered positive. Using this criterion, 96.3% of sera tested in both assays were in agreement. Doubtful results by AGID (1.7%) were clearly defined in terms of positivity and negativity by ELISA. This ELISA is suited for the rapid laboratory confirmation of AHS and should be considered as a replacement for the traditional AGID test.

African Horse Sickness↗
PubMed1989

Evidence for at least four antigenic sites on type O foot-and-mouth disease virus involved in neutralization; identification by single and multiple site monoclonal antibody-resistant mutants.

Neutralizing monoclonal antibodies raised against type O foot-and-mouth disease virus have been characterized on the basis of their reactivity with a panel of single site monoclonal antibody-resistant mutants which had defined three antigenic sites. Five antibodies neutralized all these mutants, but by selecting further single site mutants with one of these antibodies it was possible to define a fourth site involved in virus neutralization. Two monoclonal antibodies still neutralized these mutants and all multiple site resistant mutants. One multiple site resistant mutant was resistant to neutralization at each of four antigenic sites but was still efficiently neutralized by type O convalescent cattle sera. The relationship between sites recognized by different monoclonal antibodies generated in different laboratories is discussed.

Animals↗
PubMed1988

Opsonization-enhanced phagocytosis of foot-and-mouth disease virus.

Using isolated peritoneal adherent cells, in which monocytes and macrophages dominate, the uptake and destruction of foot-and-mouth disease virus (FMDV) was enhanced by the opsonization with mAb of particular epitope specificity. This was seen under conditions in which virus infectivity was not neutralized, as determined by in vitro assay. Activation of macrophages in vivo further enhanced the uptake of opsonized virus, presumably by increasing the percentage of phagocytosing cells. The enhanced phagocytosis required opsonization and apparently made use of FcR+ cells, because pepsin-treated antibodies and separated F(ab')2 fragments did not enhance the capacity of the peritoneal cells to react with the virus. The reaction also relied on active phagocytosis, because inhibition of phagocytosis using silica interfered with the binding of both virus alone and virus/antibody complexes. This evidence shows that the previous in vivo observations (McCullough et al., 1986b) of enhanced protection by the mAb can be related to active phagocytosis of virus and virus/antibody complexes. The reaction is not passive adsorption to the monocyte surface, but an active phagocytosis of the virus or the complex.

Animals↗
PubMed1987

Epitopes on foot-and-mouth disease virus particles. I. Topology.

Monoclonal antibodies (MAb) against an O1 Suisse isolate of FMDV were used to identify epitopes on the virus particle and to determine their relative function. Six major antigenic sites containing one or more epitopes were identified using competition ELISA. An epitope relationship is proposed consisting of a trypsin-sensitive sequential site, termed B2/D9, from the codings for the MAb which reacted with it, which was associated with virus infectivity and is probably at or near to the cell-binding site of the virion; a trypsin-resistant, conformational site 1C6/4C9 MAb reaction at which also resulted in neutralization of virus infectivity; a second trypsin-resistant, conformational site 3C8, where again MAb reaction neutralised virus infectivity; a third trypsin-resistant, conformational site 6C3/2G5, at which MAb-dependent neutralisation of virus infectivity was inefficient; a site 3G4, the expression of which was impaired but not destroyed by trypsin treatment, and was not related to virus infectivity; an internal site A8, which appears to be a "12S subunit-specific" site. This work clearly demonstrates for the first time that both trypsin-sensitive and trypsin-resistant neutralisable (infectivity-associated) sites exist on the FMDV particle, and only one of these can be related to the sequential site used to formulate current FMDV peptide vaccines.

Antibodies, Monoclonal↗
PubMed1987

Enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus. III. Evaluation of antibodies after infection and vaccination.

Investigations using a liquid-phase blocking sandwich enzyme-linked immunosorbent assay (ELISA) for the measurement of antibodies against foot-and-mouth disease virus (FMDV) in sera from sheep and from cattle are reported, and results compared with those obtained by virus neutralization (VN) tests. Serum antibody titres in sheep after primary vaccination and in cattle challenged with a natural aerosol after vaccination were similar by ELISA and VN. However, the antibody levels detected in sera of cattle during early infection and of vaccinated cattle after intradermolingual challenge were clearly greater by ELISA than by VN. The ELISA titres in cattle sera following synthetic peptide vaccination indicated some relationship to protection and were clearly different from those recorded by VN. On the other hand, the antibody levels following conventional vaccination showed that ELISA and VN titres in cattle sera were related to protection. Although there was a good agreement between the ELISA antibody titre and protection for the four vaccines used, by VN the titre which afforded protection varied depending on the vaccine used. The ELISA was considered therefore to be more reliable than the VN and may prove useful for evaluating the immunological response of animals following infection and following vaccination.

Animals↗
PubMed1987

Neutralization of foot-and-mouth disease virus can be mediated through any of at least three separate antigenic sites.

Seven neutralizing monoclonal antibodies were used to characterize 30 escape mutants of a type O foot-and-mouth disease (FMD) virus (O1 Kaufbeuren) selected with the five most active antibodies. Three non-overlapping antigenic sites were found by ELISA and cross-neutralization studies. Within two of the sites the epitopes of two or more monoclonal antibodies overlapped. Two of the sites were conformation-dependent and could not be detected on virus subunits or isolated denatured polypeptides. The third site was less conformation-dependent since the appropriate monoclonal antibodies were able to bind to 12S subunits, isolated VP1 protein and a synthetic peptide containing residues 141 to 160 of VP1 in ELISA. Electrofocusing of mutants of that site showed a high frequency of electrophoretic alterations in VP1. The sequence of most or all of the VP1 coding region of 10 escape mutants of that site plus three parental isolates was determined by primer extension sequencing. At least five amino acids were found to be involved but in only one case (residue 148 of VP1) did a change at that residue produce complete resistance to neutralization. Partial resistance was produced by changes at residues 144, 154 or 208 of VP1 or another residue(s), as yet undefined, that is probably in one of the other capsid polypeptides. Thus the site defined by these mutants was made up of at least three regions, the region involving residues 144 to 154 of VP1, the region encompassing residue 208 from the COOH terminus of VP1, plus a region, probably of VP2 or VP3, encompassing the undefined residue(s).

Amino Acid Sequence↗
PubMed1987

Conformational alteration in foot-and-mouth disease virus virion capsid structure after complexing with monospecific antibody.

A mechanism of neutralization of virus infectivity by antibody is described and related to the immune defences in vivo. The interaction of a particular monoclonal antibody with homologous foot-and-mouth disease virus alters the conformation of the virions to permit penetration of staining reagents. A consequence of this structural alteration is that the RNA genome becomes susceptible to dissociation from the capsid proteins. This mechanism of virus neutralization is irreversible and therefore provides an effective in vivo defence measure against virus attack, complementing the enhanced phagocytosis effected through opsonization of virions. With viruses that can replicate in phagocytes, such a mechanism of virus neutralization could provide a major 'specific' immunological defence against virus invasion.

Antibodies, Monoclonal↗
PubMed1986

A new enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus. II. Application.

The liquid-phase blocking sandwich ELISA has been evaluated for the serological study of antibodies against foot-and-mouth disease virus (FMDV). The titres recorded for sera from a population of more than 300 British uninfected, unvaccinated cattle which were examined against each of the seven immunologically distinct FMDV types were less than 1 in 40. A positive correlation between ELISA and VN titres was recorded for sera either vaccinated or involved in outbreaks of FMDV. The overall regression between the ELISA/VN data showed that 1 in 16 by VN was equivalent to 1 in 40 by ELISA. Thus ELISA is sensitive, specific and reproducible and results may be directly correlated to those recorded by VN. Serum titres may be interpreted as positive or negative and the number of sera which require retesting would be considerably less than by VN. It is suggested that this ELISA may be used as an alternative to the existing VN test for the quantification of antibodies against FMD virus.

Animals↗
PubMed1986

Use of indirect and competitive ELISAs to compare isolates of equine influenza A virus.

Antigenic differences within equine-1 and equine-2 isolates of influenza were studied by haemagglutination inhibition tests, indirect ELISA and competition ELISA, using the same antisera. Better differentiation was obtained with the competition ELISA than with the other two tests. All three methods produced similar relationships within the equine-1 isolates but differed in their ability to differentiate the equine-2 isolates where the competition ELISA was superior and produced epidemiologically sensible results. In all three tests, post-infection ferret and horse sera were more useful in discriminating isolates than post-vaccination sera.

Animals↗
PubMed1986

Immune protection against foot-and-mouth disease virus studied using virus-neutralizing and non-neutralizing concentrations of monoclonal antibodies.

Monoclonal antibodies (MAb) against sequential or conformational epitopes on foot-and-mouth disease virus (FMDV) passively protected neonatal syngeneic (BALB/c) mice at dilutions at which they could not neutralize virus infectivity in vitro. The B2, D9, 1C6 and 4C9 MAb, against the Group 1 (sequential) and Group 2 (conformational) epitopes, protected the mice at an antibody:virion molar ratio of between 38:1 and 84:1 (12-18 times lower than that required for neutralization of virus infectivity in vitro). The 3C8 (Group 3) and 6C3 (Group 4) MAb were, respectively, between 5 and 12 times, and between 18 and 40 times, less efficient at protection. There was no consistent correlation between the efficiency of neutralization of virus infectivity in vitro and the protection of neonatal mice against the virus pathogen. Thus, immune protection against FMDV must use mechanisms other than the direct neutralization of virus infectivity by antibody. Complement did not increase the virus neutralization titre of the MAb, but pepsin digestion of the MAb abrogated the enhanced in vivo protection over in vitro neutralization, with little effect on their capacity to neutralize virus infectivity. It is therefore likely that opsonization to a minimum affinity, and subsequent rapid phagocytosis, play a major role in the immune defence against FMDV. This is discussed in terms of the natural host for FMDV and the induction of immunological protection.

Animals↗
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↗
PubMed1984

Development of an enzyme-linked immunosorbent assay (ELISA) for the detection of specific antibodies against an H7N7 and an H3N8 equine influenza virus.

This paper describes a solid-phase microtitre plate enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to equine influenza viruses. Using egg-grown influenza viruses as the antigens attached to the solid phase, cross-reactions were observed between an H7N7 equine virus (designated A1) and an H3N8 equine influenza virus (designated A2) when untreated antisera were tested. Absorption of antisera with egg-grown A/Porcine/Shope/1/33 influenza virus eliminated cross-reactive antibodies so that specific detection of anti-equine influenza A1 or A2 antibodies was possible. Examination of horse sera following vaccination with A1 and/or A2 isolates showed that antibodies were produced against antigen associated with egg allantoic fluid as well as against virus. Such antibodies were eliminated following the absorption of antisera with porcine influenza virus. Results using sera from horses with known vaccination histories confirmed that the ELISA preferentially detected antibodies homologous to the antigen attached to the solid phase and methods to evaluate the current serological state of individual horses by relating the titres of specific antibodies against equine influenza A1 and A2 isolates are shown. This ELISA provides a simple and rapid method of assessing specific antibodies from horse sera and offers advantages over the 'routine' HI and SRH assessments since it gives high precision, is economical of reagents and has the capacity to handle large numbers of serum samples.

Animals↗