The objective of this study was to produce a monoclonal antibody specific for the microgametocytes of Eimeria tenella, examine the site and stage specificity of the antibody, and investigate the immunopotency of the antibody. BALB/c mice were immunized with antigen containing Eimeria tenella microgametocytes isolated from in vitro systems. After three intraperitoneal immunizations with the antigen and one booster immunization administered by tail vein injection, the mice were sacrificed and their spleen cells fused with SP2/0 mouse myeloma cells using polyethylene glycol as a fusing agent. Resultant hybridomas were screened by immunoelectrophoresis, indirect immunofluorescent antibody assay, and immunoelectron microscopy to determine the isotype, subisotype, site and stages pecificity of the antibody. Of four 96 well plates seeded with fusion products, four hybridomas were found to be producing anti body specific for the target antigen. Only the most strongly positive of these hybridomas, clone T1A3B9, was used for the study. The antibody produced by this hybridoma was found to be of sub isotype IgG2b. T1A389 monoclonal antibody was introduced into Eimeria tenella infected cell cultures on days four, five, and six post-infection. At seven days post-infection, oocyst production was assayed by fixing, staining, and counting the resultant oocysts. Results of the in vitro experiments showed a greater than 50X reduction in oocyst product ion in experimental cultures over controls. Statistical significance of the data were confirmed by a Mann-Whitney U Test. These results indicate that the monoclonal a ntibod y was exert ing an inhibitory effect on the fertilization process. T1A3B9 monoclonal antibody was incubated with Eimeria tenella infected cecal scrapings and cell culture material, immunolabeled with colloidal gold conjugates, and observed by electron microscopy. Results showed that the antibody was binding to the microgametocytes and to no other life cycle stages of the parasite, nor was it binding to host tissue. This indicates that the antibody is stage specific. Additionally, the antibody was seen to bind only to areas in close proximity to the budding flagella of developing microgametes, thus indicating distinct site specificity.

Abstract not available

<p>This study was carried out to determine the phenotype of special dog mammary gland tumours that were grown in nude mice. 26 tumours were examined by the immunohistochemical ABC-Elite protocol. The tumour tissues were labelled with following anti-human antibodies:</p><p>- AE1/AE3 (pankeratin antibody) labelled epithelial and myoepithelial cells</p><p>- CD 31 labelled endothelial cells</p><p>- desmin labelled cross-striated and smooth muscle cells</p><p>- myosin labelled cross striated muscle cells</p><p>- neurofilament (NF) labelled nerve cells</p><p>- osteopontin labelled preosteoblasts, osteoblasts and osteocytes</p><p>- p63 labelled nuclei of the myoepithelial cells</p><p>- smooth muscle actin (SMA) labelled the cytoplasm of myoepithelial cells</p><p>- type I collagen labelled the extracellular matrix in connective tissue and bone</p><p>- type II collagen labelled the extracellular matrix in cartilage</p><p>- vimentin labelled fibroblasts, fibrocytes, lipocytes, smooth muscle cells, endothelial cells, nerve cells, macrophages and myoepithelial cells</p><p>The tumours were also submitted to a double immunolabelling study using p63 and SMA.</p><p>The study could not give a final conclusion about the origin the tumours. There was still need for more research to answer that question. However, the immunohistochemical technique was analysed in detail, in order to obtain perfect labelings.</p><p>Initially, all the antibodies were tested on normal dog tissue, to acquire the best working dilutions with the lowest background problems. In the tumours, good results were obtained with these dilutions for the antibodies p63, SMA, vimentin, desmin, NF, AE1/AE3 and CD 31. Except for type I collagen, type II collagen and osteopontin that gave too much unspecific labelling of the mouse connective tissue. Even, when using the Vector® M.O.M. blocking kit, the results were still very difficult to interpretate.</p><p>The antigen retrieval methods were evaluated for all the antibodies. The antibodies p63, SMA, vimentin, desmin, AE1/AE3, myosin, neurofilament and CD 31 needed the antigen retrieval treatment. The antibodies type I collagen and type II collagen needed the treatment with the enzyme pepsin, while osteopontin did not need any pretreatment at all.</p><p>The double immunolabelling with p63 and SMA gave excellent results. Different combinations were tried out with different substrates, namely Vector® Nova RED, Vector® DAB and Vector® SG. Vector® methyl green was used as counterstaining, but it interfered with the other substrates, and better results were obtained without this counterstaining.</p>

Monoclonal antibodies were developed against USDA challenge strain of infectious laryngotracheitis virus (ILTV). Indirect immunofluorescence test was used to detect antibodies in supernatants of hybridomas. Hybridoma cells were developed by fusing Sp 2/0 myeloma cells with spleen cells obtained from mice immunized four times with partially purified USDA challenge strain of infectious laryngotracheitis virus. The supernatant of three hybridomas, designated as 2D1D8, 2E11G2, 2C6C7 were found positive for antibody activity against USDA challenge strain of ILTV. Hybridomas producing antibodies were cloned by the limiting dilution method. All three monoclonal antibodies reacted with USDA challenge strain of ILTV, S 88 00224 strain of ILTV, and 86 1169 strain of ILTV in an indirect immunofluorescence test. None of the monoclonal antibodies reacted with avian adenovirus 301 or parrot herpes virus in an indirect immunofluorescence test. The monoclonal antibodies were isotyped, and all three monoclonal antibodies were found to be IgM. / Graduation date: 1993

Humans are believed to have ~20,500 protein-coding genes andmuch effort has over the last years been put into the characterizationand localization of the encoded proteins in order to understand theirfunctions. One such effort is the Human Proteome Resource (HPR)project, started in Sweden 2003 with the aim to generate specificantibodies to each human protein and to use those antibodies toanalyze the human proteome by screening human tissues and cells.The work reported in this thesis deals with structuring of data fromantibody-based proteomics assays, with focus on the importance ofaggregating and presenting data in a way that is easy to apprehend.The goals were to model and build databases for collecting, searchingand analyzing data coming out of the large-scale HPR project and tomake all collected data publicly available. A public website, theHuman Protein Atlas, was developed giving all end-users in thescientific community access to the HPR database with proteinexpression data. In 2008, the Human Protein Atlas was released in its4th version containing more than 6000 antibodies, covering more than25% of the human proteins. All the collected protein expression datais searchable on the public website. End-users can query for proteinsthat show high expression in one tissue and no expression in anotherand possibly find tissue specific biomarkers. Queries can also beconstructed to find proteins with different expression levels in normalvs. cancer tissues. The proteins found by such a query could identifypotential biomarkers for cancer that could be used as diagnosticmarkers and maybe even be involved in cancer therapy in the future.Validation of antibodies is important in order to get reliable resultsfrom different assays. It has been noted that some antibodies arereliable in certain assays but not in others and therefore anotherpublicly available database, the Antibodypedia, has been createdwhere any antibody producer can submit their binders together withthe validation data in order for end users to purchase the bestantibody for their protein target and their intended assay. / QC 20100708

Seafood including crustaceans, on ingestion, are known to provoke gastrointestinal as well as systemic allergic reactions. Crustaceans are aquatic arthropods with a chitinous exoskeleton and include shrimp, lobster, prawn and crab. Earlier studies in our laboratory have led to the identification and characterization of three allergens from shrimp, designated as Sa-I, Sa-I1 and Sa-III. The former two were shown to be heat stable proteins with a mol. wt. of 8.4 and 34 kDa respectively, while Sa-III was identified as tRNA Arg and TRNATyr ). Sa-II was found to be the major allergen contributing to more than 50% of the allergenic activity. There are several reports on the existence of cross-reactivity among atopic allergens, in particular food allergens. It is well known that individuals with shrimp allergy often complain of adverse reactions following the ingestion of other re1ated crustaceans. Recognition of crustacea as a group causing adverse reactions in sensitive individuals has a basis in the close phylogenetic relationship of shrimp, lobster, crab and prawn. Thus, one could expect appreciable similarity in the IgE binding epitopes of the offending allergens from related crustaceans. The present study was, therefore, aimed towards the identification of the major cross-reacting crustacean allergen and localization of its IgE binding epitopes. Cross-reactivity among a1lergens from shrimp, prawn, crab and lobster was evaluated by immunochemical methods. Antigenic cross-reactivity was established by immunodiffusion using shrimp-specific rabbit IgG. Competitive ELlSA inhibition experiments using sera of shrimp sensitive patients revealed a high degree of allergenic cross-reactivity between different crustaceans. SDSPAGE and immunoblot analysis using the sera of shrimp sensitive patients have identified a 34 kDa protein as the cross-reacting crustacean allergen. Using shrimp as a model system and Sa-II as a representative crustacean allergen, further studies were carried out to get an insight into the structural and molecular basis of allergenic cross-reactivity. The strategies adopted were, (1) to raise allergen specific anti-idiotypic antibodies and explore the possibility of using these anti-idiotypic antibodies as surrogate allergens for diagnosis of crustacea allergy and (2) to identify the IgE binding epitopes on the major shrimp allergen Sa-II, which may be shared by the 34 kDa allergen from the related crustaceans. In order to explore idiotypic, anti-idiotypic and anti-anti-idiotypic responses to Sa-II, Balb/c mice were immunized with affinity purified human idiotypic antibodies directed against the purified allergen. This resulted in the production of anti-idiotypic antibodies which were quantitated using rabbit idiotypic antibodies raised against the same allergen. The mouse anti-idiotypic antibodies recognized shrimp-specific human idiotypic antibodies of the IgE isotype from 18 of 20 individuals, and IgG antibodies from 14 of 20 shrimp sensitive patients. Immunization of Balb/ c mice with affinity purified, allergen-specific anti-idiotypic antibodies induced anti-allergen IgE and IgG responses in the absence of the allergen. The induction of anti-anti-idiotypic antibodies functionally identical to allergen-specific idiotypic antibodies confirmed that the anti-idiotypic antibodies generated, are indeed a mirror image of the allergen. The present study thus provides evidence that anti-idiotypic antibodies raised against allergen-specific idiotypic antibodies may substitute for the original allergen in the induction of allergen-specific idiotypic antibodies. The demonstration of shared idiotopes on IgG and IgE antibodies in the sera of shrimp sensitive patients supports the use of allergen-specific anti-idiotypic antibodies as surrogate allergens. These anti-anti-idiotypic antibodies not only recognized Sa-II, but also the 34 kDa allergen from prawn, lobster and crab. Cross-reactivity studies using polyclonal sera of shrimp sensitive patients and Sa-II anti-anti-idiotypic antibodies have attributed the allergenic cross-reactivity observed among the related crustaceans to the presence of highly conserved IgE binding epitopes on the 34 kDa crossreacting allergen from shrimp, crab, lobster and prawn. In order to identify the igE binding epitopes on Sa-11, it was subjected to limited tryptic digestion and the peptides were separated by reverse phase HPLC. Amino acid sequence analysis of these peptides and several other peptides generated by Asp N and Lys C treatment revealed an 861 homology with the muscle protein tropomyosin from the fruit fly Drosophila melanogaster, suggesting that the major shrimp allergen is tropomyosin. To establish that Sa-II is indeed tropomyosin, the latter was isolated from shrimp and its physicochemical and immunochemical properties were compared with those of Sa-II. Both tropomyosin and Sa-II had the same molecular mass and focused in the isoelectric pH range of 4.8-5.4. In the presence of 6 M urea, the mobility of both Sa-I1 and shrimp tropomyosin shifted to give an apparent molecular mass of 50 kDa, which is a characteristic property of tropomyosins. Shrimp tropomyosin bound to specific IgE antibodies in the sera of shrimp sensitive patients as assessed by competitive ELISA inhibition and immunoblot analysis. Tropomyosin, similar to Sa-I1 was subjected to limited tryptic digestion and the tryptic maps of both Sa-II and tropomyosin as obtained by reverse phase HPLC were found to be super imposable. Dot blot immunoassay and competitive ELISA inhibition assay using the sera of shrimp sensitive patients identified two peptides, 6 and 9 that exhibited allergenic activity. Both the peptides were purified to homogeneity and sequenced. Peptide 6 is a nonapeptide corresponding to the amino adds 153-161 and peptide 9 has 17 amino acids corresponding to the aminoacid residues 50-66. The peptides individually blocked upto 50% the binding of allergen-specific IgE to hropomyosin. Sa-II specific mouse anti-anti-idiotypic antibodies recognized not only tropomyosin, but also the two allergenic peptides, thus confirming that these peptides represent the major IgE binding epitopes. The IgG binding activity was found to be associated with peptides 6 and 9 as assessed by dot blot immunoassay using the sera of shrimp sensitive patients. Thus, it was found that both IgG and IgE binding epitopes on shrimp tropomyosin are identical. Tropomyosins from both phylogenetically related and unrelated species were assessed for allergenic activity using the sera of shrimp sensitive patients. It was found that allergenic activity was associated with tropomyosins from related crustaceans and from Drosophila melanogaster which shares 86% homology with shrimp tropornyosin. However, tropomyosins from totally unrelated species like yeast, chicken, bovine, rat, rabbit and human did not exhibit allergenic activity. A comparison of the amino acid sequence of shrimp tropomyosin in the region of IgE binding epitopes with the corresponding regions of bopomyosins from different species confirmed lack of allergenic cross-reactivity. The allergenic peptides 6 and 9 were able to inhibit the binding of tropomyosins from related crustaceans to shrimp tropomyosin-specific IgE antibodies to the same extent, confirming the presence of highly conserved IgE binding epitopes. It has been established for the first time that the major crustacean allergen is the heat stable muscle protein, tropomyosin, and extensive cross-reactivity between different members of crustacea is due to the presence of highly conserved IgE binding epitopes on tropomyosins from these sources. Thus, from the present study, information with respect to the amino acid sequence of tropomyosin and localization of its 1gE binding epitopes, could be used to design synthetic peptides corresponding to the B cell and T cell epitopes which would find application in the diagnosis and desensitization of individuals allergic to crustacea.

Thesis (M.S.)--Rutgers University, 2009. / "Graduate Program in Microbiology and Molecular Genetics." Includes bibliographical references (p. 55-60).

Thesis (Ph.D.)--Drexel University, 2010. / Includes abstract and vita. Includes bibliographical references (leaves 237-258).

abstract / Microbiology / Doctoral / Doctor of Philosophy

Cancer is one of the leading causes of death worldwide. Monoclonal antibodies (mAbs) have been proved effective for cancer therapy. MAbs possess advantages over chemical drugs and small molecular drugs in cancer treatment, such as high specificity, low toxicity, effector function, long half-life in circulation system and less side effects. There are eight FDA-approved anti-cancer antibody drugs now, and many more are under development. Antibodies have two functional domains, the Fab region that is responsible for antigen recognition, and the Fc region that couples the antibody to immune effector pathways. Fab-mediated interference with cancer cell signalling may lead to growth inhibition and direct cell death, while Fc-mediated effector function through interactions with Fc-gamma receptors (FcrRs) expressed in immune cells or through complement cascades may lead to target cell cytotoxicity. Antibody engineering to increase the binding affinity and effector function may improve antibody in vivo efficacy. Anti-Her2 mAb herceptin (trastuzumab) is effective in treatment of Her2-overexpressing breast cancer patients. However, only 25–30% of patients with Her2-overexpressing tumors respond to single agent trastuzumab, and drug resistance develops even in responding patients. Accumulating evidence showed that cross-talk between Her2 and the insulin-like growth factor receptor type I (IGF-IR), including receptor heterodimerization and transactivation, and elevated IGF-IR signalling have been associated with trastuzumab resistance. Therefore, we hypothesized that dual specific antibodies co-targeting both IGF-IR and Her2 may prevent or delay the emergence of resistance to mono-specific antibodies. Mouse monoclonal antibody, M590 showed very good binding activity to IGF-IR. By engineering the CH3 domain of human Fc in pDR12 plasmid, we developed a “knobs-into-holes” hybrid IgG expression system, and successfully produced M590-Herceptin bi-specific IgG, which showed high binding avidity for both antigens and preserved antibody-dependent cell-mediated cytotoxicity (ADCC), a main route of immune protections conferred by therapeutic antibodies in vivo. M590-Herceptin dual specific antibody inhibited breast cancer and ovarian cancer cell proliferation in vitro, and inhibited cancer growth in a SKOV-3 Her2- and IGF-IR-overexpressing ovarian cancer xenograft mouse model. M590-Herceptin hybrid showed better anti-cancer activity compared with M590 and Herceptin alone, or in combination. Meantime, I also constructed a phage display antibody Fab library using the mRNA of rabbits immunized by membrane proteins of SKOV-3 cells, and isolated a novel anti-Her2 mAb, designated as 1C6. Results from in vitro assays showed that 1C6 had anti-cancer activity which was comparable to that of herceptin. M590-1C6 hybrid IgG was also constructed, and the results from in vitro assays and mouse study showed that M590-1C6 hybrid IgG also possess better inhibitory activity of Her2 positive tumours compared with m590 or 1C6 alone. In summary, this study indicates that bi-specific antibodies co-targeting two elevated cancer receptors are more effective than mono-specific antibodies for cancer therapy. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy