No description available.

No description available.

In order to develop a cheaper alternative to the conventional enzyme-linked immunosorbent assay system, application of dye molecules as labels in immunoassay was investigated in this study. This chromogenic dye-antibody conjugate could be used in colourimetric immunodetection diagnostic assays that could be used in a rural African setting. The chemistry of the interaction between twenty-six dyes of anionic, cationic and ligand dye classes with IgY and other proteins were studied for protein detection and conjugation to antibodies. Out of the twenty-six dyes studied, Direct Red 81 proved to be a good protein stain on nitrocellulose and polyacrylamide gels with comparable sensitivity to Coomassie Blue R 250. Direct Red stained proteins faster (< 5 min) than Coomassie Blue R 250 in polyacrylamide gels. Aurintricarboxylic Acid, Ethyl Red and Gallocyanine with carboxylic acid and/or hydroxyl functional groups were selected, activated with carbonyldiimidazole (CDI) to form amine reactive-imidazole intermediates and conjugated to anti-rabbit albumin IgY. Gallocyanine gave the best molar coupling ratio with IgY (76:1 dye:IgY). The dye-antibody conjugates were used to detect rabbit albumin on nitrocellulose. Aurintricarboxylic Acid-IgY and Gallocyanine-IgY detected 50 ng of rabbit albumin on nitrocellulose, which was 10 fold less sensitive than HRPO-IgY conjugate. Cross-linking of the antibodies by the dyes compromised the immunoreactivity of the Aurintricarboxylic Acid-IgY and Gallocyanine-IgY conjugates. The immunoreactivity of Ethyl Red-IgY was not compromised. Anti-rabbit albumin IgY was conjugated to derivatized dextran as an alternative immunoassay reagent and used to detect rabbit albumin on nitrocellulose by staining the polysaccharide (dextran) in the immune complex with PAS reagent. IgY-dextran complex was able to detect 25 ng of rabbit albumin on nitrocellulose, but PAS staining resulted in high background staining of the nitrocellulose membrane. Dextran-antibody conjugates may have better potential as immunodetecting reagent than dye-IgY conjugates, if a more sensitive and specific method of detecting the dextran in the Ag:Ab-dextran immune complex is developed. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

Hypermetabolism, observed in Sydenham's chorea (SC); a complication of acute rheumatoid fever (ARF) involving binding of streptococcal M protein antibodies in the brain, may result from an increase in glutamate release. The interaction of mAb 1 OF5, a specific M protein antibody subtype, with brain proteins (e.g. Rabphilin-3A), synaptic vesicles (SVs) and synaptosomal fraction (SF) was examined. Rat brain slices immunostained with mAb l OF5 revealed an interaction with choroid plexus and elements appearing to be neuropils. Dot blotting demonstrated an interaction of mAb I OF5 with both SVs and SFs. Western blotting revealed a smear from mAb 10F5 against the SF fraction. However, both modified SVs and pure liposomes examined by fluorescent and confocal microscopy bound mAbl0F5 suggesting a direct interaction with phospholipids. ELISA demonstrated binding of mAb1OF5 with negatively charged phospholipids involved in antiphospholipid syndrome (APS). Hypermetabolism and binding at the choroid plexus is observed in SC and APS supporting the connection between these disorders. / Department of Physiology and Health Science

No description available.

Mature dendritic cells (DC) isolated from lymphoid tissues initiate antigen-specific T-dependent responses even though they are non-phagocytic and weakly pinocytic, whereas Langerhans cells (LC; immature DC) can process protein antigens but are poorly immunostimulatory. Thus antigens may be acquired by cells of this lineage at an immature stage but, to our knowledge, there have been no studies on the phagocytic capacity of these cells in vitro. Using a newly-developed flow cytometric assay to measure the association between fluorescent markers and LC in epidermal cell cultures, and light and electron microscopy, we have observed phagocytosis of a variety of particles by freshly-isolated LC. The cells readily phagocytosed zymosan, heat-killed S. cerevisiae, bacteria (S. aureus and C. parvum) and fluorescent latex beads, but were unable to take up IgG- or complement-coated sheep erythrocytes, as opposed to MØ. Similarly, many freshly-isolated splenic DC had some phagocytic activity. However, the capacity of both LC and splenic DC to phagocytose zymosan, bacteria and fluorescent latex beads was markedly decreased after maturation in culture, consistently with the fact that mature DC are poorly phagocytic. Zymosan binding and uptake were much greater in fresh LC from C57BL/6 compared to BALB/c mice, and the loss of phagocytic capacity for zymosan during maturation followed different kinetics in the two strains. Two receptors mediating uptake of zymosan in LC were identified based on the effect of different inhibitors. Both of these receptors, recognising mannose and β-glucan residues, appear to be differentially regulated in the two mouse strains and during culture of LC. Our findings support the notion that DC are capable of acquiring particulate antigens for presentation at an immature stage, through recognition units for carbohydrate determinants common to a variety of potentially pathogenic organisms.

No description available.

The ability of members of the hsp70 family to bind to peptides in vivo and in vitro suggests that they may be involved in the processing of antigens for binding to Major Histocompatibility (MHC) class I and/or class n molecules. The aims of this thesis have been to provide evidence for the involvement of hsp70s in antigen processing and to characterise the binding of peptides by hspTOs by structural and functional studies. Firstly, the peptide-binding domains of two hsp70s, hsp70hom and PBP74, were expressed in isolation from the rest of the molecule for structure determination. Both of these hsp70s were implicated in antigen processing: hsp70hom in the class I pathway, due to its cytoplasmic localisation and constitutive expression, and the presence of its gene in the MHC; and PBP74 in the class n pathway because published work indicated that it was localised to endosomes and that antibodies against it inhibited antigen processing. The expression and purification of both peptide-binding domains was very successful, and one dimensional NMR experiments indicated that they were folded. However, it was not possible to determine their structures by NMR spectroscopy or X-ray crystallography because they aggregated in solution at high concentrations. Instead, the structure of the C-terminal region of hsp70hom, which includes its peptidebinding domain, was modelled based on the known structure of the equivalent portion of dnaK, the hsp70 of E.coli. The structure of hsp70hom is predicted to be very similar to that of dnaK, and modelling studies suggest that it is likely to bind peptides in a closely related fashion. The modelling of complexes between hsp70hom and two peptides suggest that the peptide-binding groove is very versatile, accounting for the broad peptide-binding specificity of hsp70s. The interactions of hsp70hom and PB74 with peptides were investigated using plate binding assays and isothermattitration calorimetry. A biotinylated peptide bound to the peptide-binding domain of hsp70hom, immobilised in plastic wells, with a Kd of <25 μM, which is within the range of Kds reported for other hsp70-peptide complexes (0.1-100 μM). In solution, isothermal titration calorimetry showed that the binding of peptides to the peptide-binding domains of hsp70hom and PBP74 was likely to be entropically rather than enthalpically driven, and, therefore, the interactions involved are likely to be predominantly hydrophobic. Secondly, PBP74, an hsp70 thought to be involved in the class II antigen processing pathway in endosomes, was localised by immunofluorescence microscopy. It was shown to be a mitochondrial protein, and is, therefore, unlikely to be involved in antigen processing. The presence of other members of the hsp70 family in lysosomes purified from a B cell line by Percoll density gradient centrifugation was investigated using antibodies that reacted with many Afferent members of the hsp70 family. No hsp70s were detected in these late endocytic compartments, even after heat shock or serum starvation. However, the presence of an hsp70 in endosomes, or of a member of this family not detected by the antibodies used, in lysosomes, cannot be ruled out. A third approach investigated the induction of the three hsp70 genes found in the MHC by four cytokines. The hsp70-l and hsp70-2 genes are induced at the mRNA level by IFN-γ and IL- 1, while TNF induces hsp70-2 alone. This data supports a role for the heat-inducible hsp70 in MHC class I antigen processing, as it appears to be coregulated with known members of this antigen processing pathway. The expression of hsp70hom was unaffected by any of the four cytokines examined. In addition, the mitochondrial hsp70 (which is not encoded in the MHC) appears to be induced by IFN-γ at the protein level. The research presented in this thesis provides a greater understanding of the peptide-binding properties of two hsp70s. Further work is necessary to show conclusively whether any of the hsp70s is involved in antigen processing.

Deficiency of the cytochrome P-450 steroid 21-hydroxylase (21-OHase) which causes Congenital Adrenal Hyperplasia (CAH) is a monogenic autosomal recessive disorder which is linked to HLA. There are two 21-OHase genes in man, A and B, and they are mapped to the HLA class III region ~ 3 kb 3' to the complement genes C4A and C4B, respectively. Two genes encoding 21-OHase were isolated, characterized and sequenced. Both 21-OHase genes are ~ 3.3 kb in length and are split into 10 exons by nine introns. Comparison of the two genes showed that although they are highly conserved, there are three deleterious mutations in the 21-OHase A gene which cause frameshifts and introduce in phase premature termination codons. Thus the 21-OHase A gene is a pseudogene. Comparison of the 21-OHase B gene to the other cytochrome P-450 sequences revealed that although the cysteine-429 was conserved in 21-OHase, there is very little homology with other cytochrome P-450, indicating it belongs to a separate family of genes within the superfamily. Clear evidence of polymorphism in 21-OHase is apparent on comparison with other 21-OHase B sequences. There is a size polymorphism of 494 and 495 amino acids. The differing severities of 21-OHase deficiency in CAH may be due to allelic variants of the 21-OHase B gene, since in most cases the defect is not due to gene deletion (Rumsby et al., 1986). A 21-OHase B gene from a patient with CAH was characterized and sequenced. There were 13 nucleotide alterations in his single 21-OHase B gene, one of which at codon 269 caused a serine to change to a threonine residue. The G → C transversion in the 21-OHase B gene from the patient at codon 269 introduced a new NcoI restriction site into the gene. This restriction fragment length polymorphism (RFLP) was used to study other patients with CAH and normal individuals. The NcoI RFLP was found not to be confined to the 21-OHase B gene but was also present in some 21-OHase A genes. It is likely therefore that the mutation occurred in the pseudogene first and then transferred to some 21-OHase B genes.

A mouse testis cDNA library was screened with an oligonucleotide probe corresponding to a sequence in the 5$ sp prime$ region of mouse N-cadherin cDNA. A composite clone containing three individual cDNAs was isolated. These included a 711 bp cDNA encoding part of mouse testicular N-cadherin, an unidentified 392 bp cDNA, and a 1500 bp cDNA encoding the mouse homologue of a human breast tumor autoantigen. / The cadherins, the influenza strain A hemagglutinins, and the fibroblast growth factor receptors are three different families of integral membrane glycoproteins that harbour the amino acid motif histidine-alanine-valine (HAV) in regions involved in protein-protein interactions. In order to identify other proteins that possess the HAV motif in functionally important regions, the SwissProt database was searched using a consensus sequence derived from the cadherins, influenza strain A hemagglutinins, and fibroblast growth factor receptors. This search identified the $ alpha$ chains of the HLA class I histocompatibility antigens as a fourth family of integral membrane glycoproteins with an HAV-containing region that is involved in a protein-protein interaction. (Abstract shortened by UMI.)