Supplemental material, sj-pdf-1-vdi-10.1177_10406387211033272 for Polyclonal antibody–based immun... more Supplemental material, sj-pdf-1-vdi-10.1177_10406387211033272 for Polyclonal antibody–based immunohistochemical detection of intraleukocytic Theileria parasites in roan and sable antelopes by Sarah J. Clift, Bernat Martí-Garcia, Rephima M. Phaswane, Emily P. Mitchell, Antoinette I. Josemans, Ilse Vorster, Katja N. Koeppel and Jeanni Fehrsen in Journal of Veterinary Diagnostic Investigation
Polyclonal antibody–based immunohistochemical detection of intraleukocytic <i>Theileria</i> parasites in roan and sable antelopes
<i>Theileria</i> parasites commonly infect African wild artiodactyls. In rare roan (&... more <i>Theileria</i> parasites commonly infect African wild artiodactyls. In rare roan (<i>Hippotragus equinus</i>) and sable (<i>H. niger</i>) antelopes, <i>Theileria</i> sp. (sable)-associated calf mortalities constrain breeding programs. The pathogenicity of most leukocyte-transforming <i>Theileria</i> spp. originates in their invasion of and multiplication in various mononuclear leukocytes, the transformation of both infected and uninfected leukocytes, and their infiltration of multiple organs. Understanding the pathogenesis of theileriosis can be improved by the use of immunohistochemistry (IHC) to identify the localization of the parasites in tissue sections. Our aim was to develop a reproducible IHC assay to detect leukocyte-associated <i>Theileria</i> parasites in formalin-fixed, paraffin-embedded roan and sable tissues. Polyclonal antibodies were purified from the sera of 5 roans from an area endemic for <i>Theileria</i> sp. (sable) and tested for IHC reactivity in 55 infected and 39 control roan and sable antelopes, and for antigen and species cross-reactivity in an additional 58 cases. The 3 strongest antibodies consistently detected intraleukocytic theilerial antigens in known positive cases in roan and sable antelopes, and also detected other <i>Theileria</i> spp. in non-hippotraginid wild artiodactyl tissues. The antibodies did not cross-react with other apicomplexan protozoa, with the exception of <i>Cryptosporidium</i>. Given that PCR on its own cannot determine the significance of theilerial infection in wild ruminants, IHC is a useful laboratory test with which to confirm the diagnosis in these species.
Recombinant antibody libraries based on chicken immunoglobulin genes are potentially valuable sou... more Recombinant antibody libraries based on chicken immunoglobulin genes are potentially valuable sources of phage displayed scFvs for use in veterinary diagnostics and research. The libraries described in this chapter are based on chicken variable heavy and light chain immunoglobulin genes joined by a short flexible linker cloned in the phagemid vector pHEN1. The resulting phagemids produce either scFvs displayed on the surface of the fusion phage subsequent to co-infection with helper phage, or soluble scFvs following IPTG induction. This chapter provides detailed and proven methods for the construction of such libraries.
A comparison of antigenic proteins in different anaplasma isolates
A Thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in f... more A Thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for a Masters degree. Onderstepoort, 1994
Onderstepoort Journal of Veterinary Research, 2017
Identifying antigenic proteins and mapping their epitopes is important for the development of dia... more Identifying antigenic proteins and mapping their epitopes is important for the development of diagnostic reagents and recombinant vaccines. B-cell epitopes of African horse sickness virus (AHSV) have previously been mapped on VP2, VP5, VP7 and NS1, using mouse, rabbit and chicken monoclonal antibodies. A comprehensive study of the humoral immune response of five vaccinated horses to AHSV-4 antigenic peptides was undertaken. A fragmented-genome phage display library expressing a repertoire of AHSV-4 peptides spanning the entire genome was constructed. The library was affinity selected for binders on immobilised polyclonal immunoglobulin G (IgG) isolated from horse sera collected pre- and post-immunisation with an attenuated AHSV-4 monovalent vaccine. The DNA inserts of binding phages were sequenced with Illumina high-throughput sequencing. The data were normalised using preimmune IgG-selected sequences. More sequences mapped to the genes coding for NS3, VP6 and VP5 than to the other ...
Polyclonal antibody–based immunohistochemical detection of intraleukocytic Theileria parasites in roan and sable antelopes
Journal of Veterinary Diagnostic Investigation
Theileria parasites commonly infect African wild artiodactyls. In rare roan ( Hippotragus equinus... more Theileria parasites commonly infect African wild artiodactyls. In rare roan ( Hippotragus equinus) and sable ( H. niger) antelopes, Theileria sp. (sable)-associated calf mortalities constrain breeding programs. The pathogenicity of most leukocyte-transforming Theileria spp. originates in their invasion of and multiplication in various mononuclear leukocytes, the transformation of both infected and uninfected leukocytes, and their infiltration of multiple organs. Understanding the pathogenesis of theileriosis can be improved by the use of immunohistochemistry (IHC) to identify the localization of the parasites in tissue sections. Our aim was to develop a reproducible IHC assay to detect leukocyte-associated Theileria parasites in formalin-fixed, paraffin-embedded roan and sable tissues. Polyclonal antibodies were purified from the sera of 5 roans from an area endemic for Theileria sp. (sable) and tested for IHC reactivity in 55 infected and 39 control roan and sable antelopes, and fo...
The antigenicity and cholesteroid nature of mycolic acids determined by recombinant chicken antib... more The antigenicity and cholesteroid nature of mycolic acids determined by recombinant chicken antibodies
Antibody fragments selected from large combinatorial libraries have numerous applications in diag... more Antibody fragments selected from large combinatorial libraries have numerous applications in diagnosis and therapy. Most existing antibody repertoires are derived from human immunoglobulin genes. Genes from other species can, however, also be used. Because of the way in which gene conversion introduces diversity, the naïve antibody repertoire of the chicken can easily be accessed using only two sets of primers. With in vitro diagnostic applications in mind, we have constructed a large library of recombinant filamentous bacteriophages displaying single chain antibody fragments derived from combinatorial pairings of chicken variable heavy and light chains. Synthetically randomised complementarity determining regions are included in some of the heavy chains. Single chain antibody fragments that recognise haptens, proteins and virus particles were selected from this repertoire. Affinities of three different antibody fragments were determined using surface plasmon resonance. Two were in ...
Cowdria ruminantium DNA is unstable in a SuperCos1 library
The Onderstepoort journal of veterinary research, 1999
A Cowdria ruminantium genomic library was constructed in a cosmid vector to serve as a source of ... more A Cowdria ruminantium genomic library was constructed in a cosmid vector to serve as a source of easily accessible and pure C. ruminantium DNA for molecular genetic studies. The cosmid library contained 846 clones which were arrayed into microtitre plates. Restriction enzyme digestion patterns indicated that these clones had an average insert size of 35 kb. Probing of the arrays did not detect any bovine clones and only one of the known C. ruminantium genes, pCS20, was detected. Due to the high AT content and the fact that C. ruminantium genes are active in the Escherichia coli host, the C. ruminantium clones were unstable in the SuperCos1 vector and most clones did not grow reproducibly. The library was contaminated with E. coli clones and these clones were maintained with greater fidelity than the C. ruminantium clones, resulting in a skewed representation over time. We have isolated seven C. ruminantium clones which we were able to serially culture reproducibly; two of these clon...
Construction and initial analysis of a representative lambda ZAPII expression library of the intracellular rickettsia Cowdria ruminantium: cloning of map1 and three other Cowdria genes
Veterinary parasitology, 1997
The causative agent of heartwater, the rickettsia Cowdria ruminantium, is very poorly understood ... more The causative agent of heartwater, the rickettsia Cowdria ruminantium, is very poorly understood at the molecular level owing to a profound lack of suitable tools. We have developed an immunoaffinity chromatographic method to purify C. ruminantium from host cell components and the purified rickettsial cells have been used to prepare substantially pure Cowdria DNA. This DNA has been used to construct what we believe to be the first fully representative C. ruminantium expression library. A clone containing the complete Cowdria map1 gene has been isolated and sequenced. This gene has been expressed in E. coli cells from the native Cowdria promoter, suggesting that the mechanisms for gene transcription and translation are similar between these two organisms. Parts of three other Cowdria genes have also been isolated and sequenced.
The ability of the Babesia equi repetitive probes, pSE2 and pSB20, to detect parasites in blood f... more The ability of the Babesia equi repetitive probes, pSE2 and pSB20, to detect parasites in blood from experimentally infected, naturally infected and carrier animals was tested using a spot hybridization assay. The clinical course of the experimentally infected horses was monitored using microscopy, indirect fluorescent antibody tests, packed cell volume, temperature and the probe assay. The probes sensitively monitored the parasite level during the development of the disease and correlated well with the other parameters tested. The sensitivity of the probe assay was superior to that of light microscopy, and a parasitaemia equivalent to less than 0.0025% could be detected. Detection orB. equi DNA was possible in all natural cases tested and 20 of the 119 randomly selected horses were identified as carriers of B. equi parasites. Microscopy could identify parasites in only 8 of these carrier animals. These results show that the probes can detect B. equi parasites in carrier animals and that they are suitable for use in a laboratory-based assay for B. equi.
Serogroup-reactive and type-specific detection of bluetongue virus antibodies using chicken scFvs in inhibition ELISAs
Journal of Virological Methods, 2005
Two bluetongue virus (BTV) serotype 10-specific single-chain Fv chicken antibody fragments (scFvs... more Two bluetongue virus (BTV) serotype 10-specific single-chain Fv chicken antibody fragments (scFvs) were evaluated in a competitive ELISA. The binding of one (F3) to purified BTV was only inhibited by antibodies against the homologous serotype. The binding of the other (F10) was blocked by antisera to each of the 24 BTV serotypes. F10 recognised VP7, a major structural protein of the BTV core, but not if the protein was directly adsorbed to a plastic surface. It did, however, bind to recombinant VP7 that had been captured from suspension by rabbit IgG. This made it possible to develop an scFv based inhibition ELISA for BTV antibodies using recombinant VP7 without prior purification. The resulting immunoassay detected antibodies to 24 BTV serotypes, but not those directed against three serotypes of the related epizootic haemorrhagic disease virus. A phage library displaying fusion peptides expressed by fragments of the BTV genome segment 7 cDNA was constructed and screened using F10. Comparing selected peptides with the amino acid sequence of VP7 showed that recognition by the scFv required at least 131 residues representing the protein&amp;amp;amp;amp;amp;amp;#39;s upper domain. By providing well-characterised immunological reagents, recombinant antibody technology can contribute to the development of improved immunoassays for BTV diagnosis.
There is an ongoing need for standardized, easily renewable immunoreagents for detecting African ... more There is an ongoing need for standardized, easily renewable immunoreagents for detecting African horsesickness virus (AHSV). Two phage displayed single-chain variable fragment (scFv) antibodies, selected from a semi-synthetic chicken antibody library, were used to develop double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to detect AHSV. In the DAS-ELISAs, the scFv previously selected with directly immobilized AHSV-3 functioned as a serotype-specific reagent that recognized only AHSV-3. In contrast, the one selected with AHSV-8 captured by IgG against AHSV-3 recognized all nine AHSV serotypes but not the Bryanston strain of equine encephalosis virus. Serving as evidence for its serogroup-specificity. These two scFvs can help to rapidly confirm the presence of AHSV while additional serotype-specific scFvs may simplify AHSV serotyping.
VP7, the major structural core protein of bluetongue virus, is conserved among the 24 BTV serotyp... more VP7, the major structural core protein of bluetongue virus, is conserved among the 24 BTV serotypes. The gene encoding VP7 of serotype 4 was expressed in E. coli. A semisynthetic chicken antibody library was screened with the resulting protein. Six singlechain antibody fragments (scFvs) were isolated. Immune sera blocked the binding of four of the six scFvs in ELISA. These scFvs recognised recombinant VP7 coated directly onto a plastic surface. Their behaviour therefore differs from that of scFv F10 (Fehrsen et al., 2005) which was selected earlier on directly immobilised bluetongue virus and which binds to VP7 only when it is captured by an immobilised immunoglobulin directed against bluetongue virus.
The development of new and effective vaccines and immunodiagnostic reagents requires the characte... more The development of new and effective vaccines and immunodiagnostic reagents requires the characterisation of antigenically relevant proteins and their interactions with the products of the immune system. Phage display technology was investigated as a means of elucidating some of the antigenic properties of the rickettsial parasite, Cowdria ruminantium (Cowdria). Randomly fragmented gene-derived libraries have been useful in elucidating viral and other epitopes, but only limited work has been done with entire genomes. A phage display library expressing a repertoire of Cowdria peptides was constructed. It was sufficiently large to represent the organism's genome, but lacked phages displaying peptides coded for by genes containing a Pvu II restriction enzyme site, including the one coding for the major antigenic protein 1 (MAP1). This was considered advantageous since MAP1 is immunodominant and has already been well characterised. Affinity selection with antibodies against Cowdria proteins other than MAP1 allowed several antibodyreactive peptides to be isolated. These selected sequences were placed in the context of the genome by screening a lambda bacteriophage library and by comparison with Cowdria DNA sequences. Apart from showing that antigenic mimics were present in the phage display library, six open reading frames encoding putative Cowdria proteins were identified. All had similarities to, or motifs in common with, membrane proteins and are thus likely to be exposed to the host's humoral immune system. Some of the proteins identified were larger than the antigens used to elicit the antibodies used for selection, probably as a result of the presence of cross-reactive epitopes. Despite limitations experienced when extending a fragmented-gene approach for epitope location to genomes, it was possible to identify an antigenic region on MAP1 by comparison with selected mimics. In addition, binding peptide sequences were identified with two monoclonal antibodies that had been raised against non-Cowdria antigens. An epitope on the VP7 protein of bluetongue virus was identified and peptides were found that reacted with a monoclonal antibody directed against iii malignant catarrhal fever virus. Thus, apart from being able to identify several potentially important Cowdria epitopes and genes, the fragmented-genome library holds promise as a universal reagent for identifying useful mimics. iv
Two chicken single-chain variable region antibody fragments (scFvs) that recognised the 65 kDa he... more Two chicken single-chain variable region antibody fragments (scFvs) that recognised the 65 kDa heatshock protein (HSP65) of Mycobacterium bovis were selected from a large semi-synthetic phage displayed library. Both recognised HSP65 in indirect enzyme-linked immunosorbent assay (ELISA) and immunoblots and retained their activity during storage. Neither, however, could function as the capture reagent in a sandwich ELISA when immobilised on polystyrene. To establish whether they could be engineered for general use in immunotests, the genes coding for these scFvs were subcloned in expression vectors that contained sequences encoding chicken IgY heavy-chain constant region domains. This resulted in larger bivalent constructs which more closely resembled IgY molecules. The engineered fragments were evaluated in ELISAs and gold-conjugated immunochromatographic tests (ICTs). In contrast to their previous behaviour as scFvs, the modified fragments (designated ''gallibodies'') could be used for immunocapture in ELISA and could be readily conjugated to colloidal gold nanoparticles. A sandwich ICT that could detect recombinant HSP65 was also devised. Although converting the recombinant single-chain monomeric antibody fragments to bivalent immunoglobulin-like molecules did not entirely 'standardise' the behaviour of the scFvs, this approach remains potentially useful for developing practical, robust, immunodiagnostic reagents.
Recombinant antibodies can be engineered to improve their binding or other characteristics. A chi... more Recombinant antibodies can be engineered to improve their binding or other characteristics. A chicken single chain variable fragment (scFv) phage display library was panned against the mycobacterial 16 kDa antigen. Three fusion phages which bound specifically to the antigen were selected, each of which produced low signals in ELISA when secreted as a soluble scFv. One scFv was therefore chosen to be modified in an attempt to improve its binding. Firstly, a mutant sublibrary was created by random mutagenesis. High stringency panning of this sublibrary yielded binders which produced ELISA signals up to eleven times higher than the parent scFv. An increase in the intrinsic affinity was confirmed by surface plasmon resonance. Secondly, the flexible linker between the heavy and light chains of the parent scFv was either shortened to one glycine residue or deleted entirely. No ELISA signal was obtained when the linker was absent, but the glycine-linked scFv showed enhanced binding. Size exclusion chromatography revealed that the enhanced binder had aggregated to form tetramers. This study confirms that the strategies used to improve the binding of human and mouse scFvs can also enhance chicken scFvs.
Supplemental material, sj-pdf-1-vdi-10.1177_10406387211033272 for Polyclonal antibody–based immun... more Supplemental material, sj-pdf-1-vdi-10.1177_10406387211033272 for Polyclonal antibody–based immunohistochemical detection of intraleukocytic Theileria parasites in roan and sable antelopes by Sarah J. Clift, Bernat Martí-Garcia, Rephima M. Phaswane, Emily P. Mitchell, Antoinette I. Josemans, Ilse Vorster, Katja N. Koeppel and Jeanni Fehrsen in Journal of Veterinary Diagnostic Investigation
Polyclonal antibody–based immunohistochemical detection of intraleukocytic <i>Theileria</i> parasites in roan and sable antelopes
<i>Theileria</i> parasites commonly infect African wild artiodactyls. In rare roan (&... more <i>Theileria</i> parasites commonly infect African wild artiodactyls. In rare roan (<i>Hippotragus equinus</i>) and sable (<i>H. niger</i>) antelopes, <i>Theileria</i> sp. (sable)-associated calf mortalities constrain breeding programs. The pathogenicity of most leukocyte-transforming <i>Theileria</i> spp. originates in their invasion of and multiplication in various mononuclear leukocytes, the transformation of both infected and uninfected leukocytes, and their infiltration of multiple organs. Understanding the pathogenesis of theileriosis can be improved by the use of immunohistochemistry (IHC) to identify the localization of the parasites in tissue sections. Our aim was to develop a reproducible IHC assay to detect leukocyte-associated <i>Theileria</i> parasites in formalin-fixed, paraffin-embedded roan and sable tissues. Polyclonal antibodies were purified from the sera of 5 roans from an area endemic for <i>Theileria</i> sp. (sable) and tested for IHC reactivity in 55 infected and 39 control roan and sable antelopes, and for antigen and species cross-reactivity in an additional 58 cases. The 3 strongest antibodies consistently detected intraleukocytic theilerial antigens in known positive cases in roan and sable antelopes, and also detected other <i>Theileria</i> spp. in non-hippotraginid wild artiodactyl tissues. The antibodies did not cross-react with other apicomplexan protozoa, with the exception of <i>Cryptosporidium</i>. Given that PCR on its own cannot determine the significance of theilerial infection in wild ruminants, IHC is a useful laboratory test with which to confirm the diagnosis in these species.
Recombinant antibody libraries based on chicken immunoglobulin genes are potentially valuable sou... more Recombinant antibody libraries based on chicken immunoglobulin genes are potentially valuable sources of phage displayed scFvs for use in veterinary diagnostics and research. The libraries described in this chapter are based on chicken variable heavy and light chain immunoglobulin genes joined by a short flexible linker cloned in the phagemid vector pHEN1. The resulting phagemids produce either scFvs displayed on the surface of the fusion phage subsequent to co-infection with helper phage, or soluble scFvs following IPTG induction. This chapter provides detailed and proven methods for the construction of such libraries.
A comparison of antigenic proteins in different anaplasma isolates
A Thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in f... more A Thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for a Masters degree. Onderstepoort, 1994
Onderstepoort Journal of Veterinary Research, 2017
Identifying antigenic proteins and mapping their epitopes is important for the development of dia... more Identifying antigenic proteins and mapping their epitopes is important for the development of diagnostic reagents and recombinant vaccines. B-cell epitopes of African horse sickness virus (AHSV) have previously been mapped on VP2, VP5, VP7 and NS1, using mouse, rabbit and chicken monoclonal antibodies. A comprehensive study of the humoral immune response of five vaccinated horses to AHSV-4 antigenic peptides was undertaken. A fragmented-genome phage display library expressing a repertoire of AHSV-4 peptides spanning the entire genome was constructed. The library was affinity selected for binders on immobilised polyclonal immunoglobulin G (IgG) isolated from horse sera collected pre- and post-immunisation with an attenuated AHSV-4 monovalent vaccine. The DNA inserts of binding phages were sequenced with Illumina high-throughput sequencing. The data were normalised using preimmune IgG-selected sequences. More sequences mapped to the genes coding for NS3, VP6 and VP5 than to the other ...
Polyclonal antibody–based immunohistochemical detection of intraleukocytic Theileria parasites in roan and sable antelopes
Journal of Veterinary Diagnostic Investigation
Theileria parasites commonly infect African wild artiodactyls. In rare roan ( Hippotragus equinus... more Theileria parasites commonly infect African wild artiodactyls. In rare roan ( Hippotragus equinus) and sable ( H. niger) antelopes, Theileria sp. (sable)-associated calf mortalities constrain breeding programs. The pathogenicity of most leukocyte-transforming Theileria spp. originates in their invasion of and multiplication in various mononuclear leukocytes, the transformation of both infected and uninfected leukocytes, and their infiltration of multiple organs. Understanding the pathogenesis of theileriosis can be improved by the use of immunohistochemistry (IHC) to identify the localization of the parasites in tissue sections. Our aim was to develop a reproducible IHC assay to detect leukocyte-associated Theileria parasites in formalin-fixed, paraffin-embedded roan and sable tissues. Polyclonal antibodies were purified from the sera of 5 roans from an area endemic for Theileria sp. (sable) and tested for IHC reactivity in 55 infected and 39 control roan and sable antelopes, and fo...
The antigenicity and cholesteroid nature of mycolic acids determined by recombinant chicken antib... more The antigenicity and cholesteroid nature of mycolic acids determined by recombinant chicken antibodies
Antibody fragments selected from large combinatorial libraries have numerous applications in diag... more Antibody fragments selected from large combinatorial libraries have numerous applications in diagnosis and therapy. Most existing antibody repertoires are derived from human immunoglobulin genes. Genes from other species can, however, also be used. Because of the way in which gene conversion introduces diversity, the naïve antibody repertoire of the chicken can easily be accessed using only two sets of primers. With in vitro diagnostic applications in mind, we have constructed a large library of recombinant filamentous bacteriophages displaying single chain antibody fragments derived from combinatorial pairings of chicken variable heavy and light chains. Synthetically randomised complementarity determining regions are included in some of the heavy chains. Single chain antibody fragments that recognise haptens, proteins and virus particles were selected from this repertoire. Affinities of three different antibody fragments were determined using surface plasmon resonance. Two were in ...
Cowdria ruminantium DNA is unstable in a SuperCos1 library
The Onderstepoort journal of veterinary research, 1999
A Cowdria ruminantium genomic library was constructed in a cosmid vector to serve as a source of ... more A Cowdria ruminantium genomic library was constructed in a cosmid vector to serve as a source of easily accessible and pure C. ruminantium DNA for molecular genetic studies. The cosmid library contained 846 clones which were arrayed into microtitre plates. Restriction enzyme digestion patterns indicated that these clones had an average insert size of 35 kb. Probing of the arrays did not detect any bovine clones and only one of the known C. ruminantium genes, pCS20, was detected. Due to the high AT content and the fact that C. ruminantium genes are active in the Escherichia coli host, the C. ruminantium clones were unstable in the SuperCos1 vector and most clones did not grow reproducibly. The library was contaminated with E. coli clones and these clones were maintained with greater fidelity than the C. ruminantium clones, resulting in a skewed representation over time. We have isolated seven C. ruminantium clones which we were able to serially culture reproducibly; two of these clon...
Construction and initial analysis of a representative lambda ZAPII expression library of the intracellular rickettsia Cowdria ruminantium: cloning of map1 and three other Cowdria genes
Veterinary parasitology, 1997
The causative agent of heartwater, the rickettsia Cowdria ruminantium, is very poorly understood ... more The causative agent of heartwater, the rickettsia Cowdria ruminantium, is very poorly understood at the molecular level owing to a profound lack of suitable tools. We have developed an immunoaffinity chromatographic method to purify C. ruminantium from host cell components and the purified rickettsial cells have been used to prepare substantially pure Cowdria DNA. This DNA has been used to construct what we believe to be the first fully representative C. ruminantium expression library. A clone containing the complete Cowdria map1 gene has been isolated and sequenced. This gene has been expressed in E. coli cells from the native Cowdria promoter, suggesting that the mechanisms for gene transcription and translation are similar between these two organisms. Parts of three other Cowdria genes have also been isolated and sequenced.
The ability of the Babesia equi repetitive probes, pSE2 and pSB20, to detect parasites in blood f... more The ability of the Babesia equi repetitive probes, pSE2 and pSB20, to detect parasites in blood from experimentally infected, naturally infected and carrier animals was tested using a spot hybridization assay. The clinical course of the experimentally infected horses was monitored using microscopy, indirect fluorescent antibody tests, packed cell volume, temperature and the probe assay. The probes sensitively monitored the parasite level during the development of the disease and correlated well with the other parameters tested. The sensitivity of the probe assay was superior to that of light microscopy, and a parasitaemia equivalent to less than 0.0025% could be detected. Detection orB. equi DNA was possible in all natural cases tested and 20 of the 119 randomly selected horses were identified as carriers of B. equi parasites. Microscopy could identify parasites in only 8 of these carrier animals. These results show that the probes can detect B. equi parasites in carrier animals and that they are suitable for use in a laboratory-based assay for B. equi.
Serogroup-reactive and type-specific detection of bluetongue virus antibodies using chicken scFvs in inhibition ELISAs
Journal of Virological Methods, 2005
Two bluetongue virus (BTV) serotype 10-specific single-chain Fv chicken antibody fragments (scFvs... more Two bluetongue virus (BTV) serotype 10-specific single-chain Fv chicken antibody fragments (scFvs) were evaluated in a competitive ELISA. The binding of one (F3) to purified BTV was only inhibited by antibodies against the homologous serotype. The binding of the other (F10) was blocked by antisera to each of the 24 BTV serotypes. F10 recognised VP7, a major structural protein of the BTV core, but not if the protein was directly adsorbed to a plastic surface. It did, however, bind to recombinant VP7 that had been captured from suspension by rabbit IgG. This made it possible to develop an scFv based inhibition ELISA for BTV antibodies using recombinant VP7 without prior purification. The resulting immunoassay detected antibodies to 24 BTV serotypes, but not those directed against three serotypes of the related epizootic haemorrhagic disease virus. A phage library displaying fusion peptides expressed by fragments of the BTV genome segment 7 cDNA was constructed and screened using F10. Comparing selected peptides with the amino acid sequence of VP7 showed that recognition by the scFv required at least 131 residues representing the protein&amp;amp;amp;amp;amp;amp;#39;s upper domain. By providing well-characterised immunological reagents, recombinant antibody technology can contribute to the development of improved immunoassays for BTV diagnosis.
There is an ongoing need for standardized, easily renewable immunoreagents for detecting African ... more There is an ongoing need for standardized, easily renewable immunoreagents for detecting African horsesickness virus (AHSV). Two phage displayed single-chain variable fragment (scFv) antibodies, selected from a semi-synthetic chicken antibody library, were used to develop double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to detect AHSV. In the DAS-ELISAs, the scFv previously selected with directly immobilized AHSV-3 functioned as a serotype-specific reagent that recognized only AHSV-3. In contrast, the one selected with AHSV-8 captured by IgG against AHSV-3 recognized all nine AHSV serotypes but not the Bryanston strain of equine encephalosis virus. Serving as evidence for its serogroup-specificity. These two scFvs can help to rapidly confirm the presence of AHSV while additional serotype-specific scFvs may simplify AHSV serotyping.
VP7, the major structural core protein of bluetongue virus, is conserved among the 24 BTV serotyp... more VP7, the major structural core protein of bluetongue virus, is conserved among the 24 BTV serotypes. The gene encoding VP7 of serotype 4 was expressed in E. coli. A semisynthetic chicken antibody library was screened with the resulting protein. Six singlechain antibody fragments (scFvs) were isolated. Immune sera blocked the binding of four of the six scFvs in ELISA. These scFvs recognised recombinant VP7 coated directly onto a plastic surface. Their behaviour therefore differs from that of scFv F10 (Fehrsen et al., 2005) which was selected earlier on directly immobilised bluetongue virus and which binds to VP7 only when it is captured by an immobilised immunoglobulin directed against bluetongue virus.
The development of new and effective vaccines and immunodiagnostic reagents requires the characte... more The development of new and effective vaccines and immunodiagnostic reagents requires the characterisation of antigenically relevant proteins and their interactions with the products of the immune system. Phage display technology was investigated as a means of elucidating some of the antigenic properties of the rickettsial parasite, Cowdria ruminantium (Cowdria). Randomly fragmented gene-derived libraries have been useful in elucidating viral and other epitopes, but only limited work has been done with entire genomes. A phage display library expressing a repertoire of Cowdria peptides was constructed. It was sufficiently large to represent the organism's genome, but lacked phages displaying peptides coded for by genes containing a Pvu II restriction enzyme site, including the one coding for the major antigenic protein 1 (MAP1). This was considered advantageous since MAP1 is immunodominant and has already been well characterised. Affinity selection with antibodies against Cowdria proteins other than MAP1 allowed several antibodyreactive peptides to be isolated. These selected sequences were placed in the context of the genome by screening a lambda bacteriophage library and by comparison with Cowdria DNA sequences. Apart from showing that antigenic mimics were present in the phage display library, six open reading frames encoding putative Cowdria proteins were identified. All had similarities to, or motifs in common with, membrane proteins and are thus likely to be exposed to the host's humoral immune system. Some of the proteins identified were larger than the antigens used to elicit the antibodies used for selection, probably as a result of the presence of cross-reactive epitopes. Despite limitations experienced when extending a fragmented-gene approach for epitope location to genomes, it was possible to identify an antigenic region on MAP1 by comparison with selected mimics. In addition, binding peptide sequences were identified with two monoclonal antibodies that had been raised against non-Cowdria antigens. An epitope on the VP7 protein of bluetongue virus was identified and peptides were found that reacted with a monoclonal antibody directed against iii malignant catarrhal fever virus. Thus, apart from being able to identify several potentially important Cowdria epitopes and genes, the fragmented-genome library holds promise as a universal reagent for identifying useful mimics. iv
Two chicken single-chain variable region antibody fragments (scFvs) that recognised the 65 kDa he... more Two chicken single-chain variable region antibody fragments (scFvs) that recognised the 65 kDa heatshock protein (HSP65) of Mycobacterium bovis were selected from a large semi-synthetic phage displayed library. Both recognised HSP65 in indirect enzyme-linked immunosorbent assay (ELISA) and immunoblots and retained their activity during storage. Neither, however, could function as the capture reagent in a sandwich ELISA when immobilised on polystyrene. To establish whether they could be engineered for general use in immunotests, the genes coding for these scFvs were subcloned in expression vectors that contained sequences encoding chicken IgY heavy-chain constant region domains. This resulted in larger bivalent constructs which more closely resembled IgY molecules. The engineered fragments were evaluated in ELISAs and gold-conjugated immunochromatographic tests (ICTs). In contrast to their previous behaviour as scFvs, the modified fragments (designated ''gallibodies'') could be used for immunocapture in ELISA and could be readily conjugated to colloidal gold nanoparticles. A sandwich ICT that could detect recombinant HSP65 was also devised. Although converting the recombinant single-chain monomeric antibody fragments to bivalent immunoglobulin-like molecules did not entirely 'standardise' the behaviour of the scFvs, this approach remains potentially useful for developing practical, robust, immunodiagnostic reagents.
Recombinant antibodies can be engineered to improve their binding or other characteristics. A chi... more Recombinant antibodies can be engineered to improve their binding or other characteristics. A chicken single chain variable fragment (scFv) phage display library was panned against the mycobacterial 16 kDa antigen. Three fusion phages which bound specifically to the antigen were selected, each of which produced low signals in ELISA when secreted as a soluble scFv. One scFv was therefore chosen to be modified in an attempt to improve its binding. Firstly, a mutant sublibrary was created by random mutagenesis. High stringency panning of this sublibrary yielded binders which produced ELISA signals up to eleven times higher than the parent scFv. An increase in the intrinsic affinity was confirmed by surface plasmon resonance. Secondly, the flexible linker between the heavy and light chains of the parent scFv was either shortened to one glycine residue or deleted entirely. No ELISA signal was obtained when the linker was absent, but the glycine-linked scFv showed enhanced binding. Size exclusion chromatography revealed that the enhanced binder had aggregated to form tetramers. This study confirms that the strategies used to improve the binding of human and mouse scFvs can also enhance chicken scFvs.
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Papers by Jeanni Fehrsen