A serological study of some cauliflower mosaic virus isolates

Bibliography: pages 134-149.Enzyme-linked immunosorbent assay (ELISA) was used successfully to detect cauliflower mosaic virus (CaMV) in crude leaf extracts. Small serological differences between CaMV isolates could be shown by ELISA and serum cross-absorption. Serological reactivity of CaMV was found to depend on the proteolytic degradation state of the virus coat protein so making it impossible to establish definite serological relationships among the virus isolates tested. Proteolysis during purification of CaMV could not be entirely eliminated. The coat protein of CaMV was shown to be glycosylated by the specific binding of labelled Concanavalin A. The role of carbohydrate residues in CaMV serological reactivity was evaluated

Identification of genes coding for B cell antigens of Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC) by using phage display

<p>Abstract</p> <p>Background</p> <p>Contagious bovine pleuropneumonia (CBPP) is a mycoplasmal disease caused by <it>Mycoplasma mycoides </it>subsp. <it>mycoides </it>SC (<it>Mmm</it>SC). Since the disease is a serious problem that can affect cattle production in parts of Africa, there is a need for an effective and economical vaccine. Identifying which of the causative agent's proteins trigger potentially protective immune responses is an important step towards developing a subunit vaccine. Accordingly, the purpose of this study was to determine whether phage display combined with bioinformatics could be used to narrow the search for genes that code for potentially immunogenic proteins of <it>Mmm</it>SC. Since the production of IgG2 and IgA are associated with a Th₁cellular immune response which is implicated in protection against CBPP, antigens which elicit these immunoglobulin subclasses may be useful in developing a subunit vaccine.</p> <p>Results</p> <p>A filamentous phage library displaying a repertoire of peptides expressed by fragments of the genome of <it>MmmSC </it>was constructed. It was subjected to selection using antibodies from naturally- and experimentally-infected cattle. Mycoplasmal genes were identified by matching the nucleotide sequences of DNA from immunoselected phage particles with the mycoplasmal genome. This allowed a catalogue of genes coding for the proteins that elicited an immune response to be compiled. Using this method together with computer algorithms designed to score parameters that influence surface accessibility and hence potential antigenicity, five genes (<it>abc, gapN, glpO, lppB </it>and <it>ptsG</it>) were chosen to be expressed in <it>Escherichia coli</it>. After appropriate site-directed mutagenesis, polypeptides representing portions of each of these proteins were tested for immunoreactivity. Of these five, polypeptides representing expression products of <it>abc </it>and <it>lppB </it>were recognised on immunoblots by sera obtained from cattle during a natural outbreak of the disease.</p> <p>Conclusion</p> <p>Since phage display physically couples phenotype with genotype, it was used to compile a list of sequences that code for <it>Mmm</it>SC proteins bearing epitopes which were recognised by antibodies in the serum of infected animals. Together with the appropriate bioinformatic analyses, this approach provided several potentially useful vaccine or diagnostic leads. The phage display step empirically identified sequences by their interaction with antibodies which accordingly reduced the number of ORFs that had to be expressed for testing. This is a particular advantage when working with <it>Mmm</it>SC since the mycoplasmal codon for tryptophan needs to be mutated to prevent it from being translated as a stop in <it>E. coli</it>.</p

Immunoassays with chemically modified bacteriophage

Bibliography: pages 163-176.The immunospecific inactivation of bacteriophage is one of the most sensitive methods available for the detection of very low concentrations of antibody. By chemically modifying the phage coat-protein, this sensitivity can be extended to antibodies against a wide variety of haptens and proteins. Phage particles that have been modified by attaching some molecule onto their surface are sensitive to antibodies directed against the coupled chemical moiety. Furthermore, the inactivation of the modified phage by antibody can be inhibited by free antigen, and this provides a sensitive assay for small quantities of antigen. Antibody and antigens have been detected at the nanogram level by this technique. The modified phage technique can also be used to distinguish antibodies of different specificities and to discriminate between closely related antigens. This technique has not yet been applied to the immunochemical study of viral components and the present work represents such an attempt. Tobacco mosaic virus (TMV) was chosen as a model system since it permits the study of numerous inununological phenomena (Rappaport, 1965; van Regenmortel, 1966). A series of preliminary experiments were performed to obtain experience in the methodology of the technique. These included the inactivation of native T4 phage by phage antiserum and anti phage IgG, the chemical modification of phage with DNP and the attachment of lysozyme to phage under a variety of conditions. The success of the covalent binding of protein to phage particles depends on finding conditions under which a proportion of the phage remains viable and, at the same time, can still be neutralized by anti-protein sera. To this end, different proportions of reactants and three different bifunctional reagents were tested. To prevent aggregation of tobacco mosaic virus protein (TMVP) at the high concentration used, the protein was treated with N-bromosuccinimide. TMVP-phage conjugates which were sensitive to antiserum were prepared using bis-diazobenzidine as the bifunctional reagent

Chicken scFvs and bivalent scFv-CH fusions directed against HSP65 of Mycobacterium bovis

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

Improving the characteristics of a mycobacterial 16 kDa-specific chicken scFv

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 antigenwere 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

History of medicine : the Hamilton Naki Clinical Scholarship, 2007-2011

The original publication is available at http://www.samj.org.zaThe Hamilton Naki Scholarship was introduced because of the shortage of qualified academic leaders in South African medical schools, especially for academic clinicians from previously disadvantaged backgrounds. There were only a handful of African academic doctors with a significant published record of scholarship in South Africa. If academic physicians from the whole population were not recruited and trained, South Africa would lose its ability to train high-quality health practitioners. To address these deficiencies, the Netcare Physician Partnerships Trust established a scholarship to produce world-class academics in all medical specialties to teach and conduct research comparable to other parts of the world