No description available.

This study focuses on the utilization of metal-dependent monoclonal antibodies for large-scale chromatography and addresses an aspect that has been cited to lower immunosorbent performance, namely "orientation" of antibodies on matrices. The antibodies used in this study, the "EDTAdependent" 7D7BlO and the "Ca²⁺ -dependent" HPC4 are directed against human Protein C (PC). The 7D7BI0 antibody was characterized in terms of its metaldependency and specificity. The region of PC (epitope) recognized by 7D7BlO was identified as the first 15 residues in the NH₂-terminal. Immunosorbents made with 7D7BI0 provided highly pure and functional PC. The "orientation" of the antibodies on matrices was addressed in two ways. In the first approach, performance of immunosorbents coupled through carbohydrate moieties were compared with immunosorbents coupled through peptide regions. Coupling via carbohydrate linkages, which is generally believed to be Fc-directed, did not have any advantage in terms of efficiency and recovery over coupling via peptide. / Ph. D.

Four macrophage (Mφ) surface antigens (Ia, Mac-1, -2, and -3) were examined for their association with Mφ regulatory functions. Observations of antigen expression on Mφ derived from normal or tumor-bearing hosts (TBH) showed that changes occurred in the antigen-defined phenotypes of Mφ which evolve during tumor growth. These changes in antigen expression were correlated with notable changes in Mφ immunoregulatory functions. Experiments using only normal host-derived Mφ showed that in the presence of complement (C), monoclonal antibodies (mAb) directed against Mφ could lyse targeted Mφ and that enrichment of the remaining cells provided populations of Mφ that were altered in their regulatory functions. Analysis of mAb-treated Mφ in the absence of C, suggested that the alterations observed in the presence of C were not due to ligand-receptor activation of peritoneal Mφ and that antibodies alone were not altering Mφ viability. When anti-Mac- I, -2, and -3 antibodies, were used to modify accessory cell activity of whole spleen cell (WSC) or splenic adherent cell (SAC) preparations from normal or TBII, differential susceptibilities of the Mφ were noted. Ligand-receptor activation of WSC by anti-Mac-I was observed in normal but not TBH WSC. With C, anti-Mac-I and -3 each reduced normal and TBH WSC proliferation. To evaluate the possible role of different types of SAC in T cell lectin responsiveness, adherent cells were collected and depleted by antibody plus C treatment and added back to normal T cells. Removal of Mac-1⁺ normal host SAC stimulated the supportive accessory function of the remaining SAC. Enhancing accessory cell function diminished after removal of normal host Mac-2⁺ or TBH Mac-1⁺ SAC. In summary, SAC from normal host demonstrated an accessory cell function corresponding to a Mac-1⁻ phenotype, which was either replaced or obscured by the predominance of a Mac-1⁺ phenotype in TBH. Variable Ia antigen expression by Mφ was examined during tumor growth. Tumor growth induced progressive loss of Ia antigen expression on Mφ. TBH splenic Mφ supported Concanavalin A-induced proliferation of syngeneic T cells (Ia antigen-independent) but did not support syngeneic T cell proliferation in the mixed lymphocyte reaction (MLR) (Ia antigen-dependent). Irrespective of tissue source, normal and TBH Mφ differed in their MLR stimulatory capabilities. In general, splenic Mφ preparations were better stimulators of allogeneic T cell blastogenesis in the MLR than thioglycollate-elicited peritoneal Mφ. Expression of Ia antigens by normal but not TBH Mφ were diminished by 24-hr in vitro plating of the peritoneal Mφ. Indomethacin treatment showed Prostaglandin E₂ was not a direct in vitro factor in Ia antigen-mediated reduction of splenic Mφ MLR stimulatory activity. Taken together, this data suggested a loss of Mφ Ia antigen expression, resulting in a decrease in Ia antigen-mediated functional activities during tumor growth. To continue the assessment of Mφ phenotypes and to determine if alterations in Mφ function during tumor growth included changes in the secretion of soluble regulators of T cell activities, anti-Mac-1, -2, and -3 mAb were used to modulate monokine-mediated regulation of T lymphocyte proliferation. The mAb anti-Mac-1, -2, and -3 (plus C) exhibited differential depletion of normal and TBH Mφ. There was a distinct increase in the number of peroxidase-positive Mφ during tumor growth. Peroxidase-positive TBH Mφ were susceptible to C-mediated lysis by anti-Mac-1 and -3 but not anti-Mac-2, whereas no direct relationship was observed among normal host-derived Mφ. Immunofluorescence of mAb-binding showed a decrease in Mac-2⁺ cells in TBH Mφ populations that was accompanied by an increase in Mac-3 expression. Anti-Mac-2 treatment significantly reduced the ability of TBH Mφ to produce a soluble suppressor(s) but did not alter normal host Mφ-derived suppressor production. In contrast, anti-Mac-1 and -3 treatment of normal host Mφ significantly reduced suppressor production but had diminished effects on TBH Mφ. Anti-Ia plus C treatment of splenic or peritoneal Mφ derived from normal or TBH showed that selection of Ia Mφ increased the secretion of PGE and also increased the T cell suppressor activity in Mφ culture supernatants. Collectively, these data suggest that tumor-induced aberrations in immunoregulation can be attributed to differences in Mφ subpopulations which were discriminated by their surface membrane components. / Ph. D.

Atlantic cod (Gadus morhua L.) is one of the most promising new fish species introduced to cold water aquaculture due to the large established market in Europe and the USA and the decline in wild stock. So far, the production of farmed cod has been relatively low, with the main hindrance due to diseases. Vibrio anguillarum has been recognised as the biggest disease problem of farmed cod and has slowed the development of a successful cod aquaculture industry. When the first incidences of V. anguillarum occurred in cod aquaculture, vaccines designed for vibriosis in Atlantic salmon (Salmo salar L.) were used in an attempt to combat the disease. However, these vaccines did not provide sufficient protection, possibly because they lacked serotype O2b, which is known to affect cod and to a lesser extent salmonids. Recently, vibriosis vaccines specifically designed to protect Atlantic cod have been formulated, but outbreaks of vibriosis in vaccinated fish are still being reported, suggesting that these formulations are inadequate. The aim of this project was to develop a whole cell inactivated vaccine formulation specifically tailored to protect Atlantic cod against Vibrio anguillarum. The serological classification of V. anguillarum was first investigated by producing a set of monoclonal antibodies (mAbs). Using lipopolysaccharides (LPS) extracted with butan-1-ol, 4 mAbs were selected and shown to react specifically with V. anguillarum serotypes O1, O2a and O2b. A collection of over 150 V. anguillarum isolates were screened using these, which revealed that most of the isolates had been previously correctly classified. A new sub-serotype of V. anguillarum O2 was identified from isolates recovered from outbreaks of vibriosis in Norway as well as Scotland. This new sub-serotype was referred to as O2d since the subserotype O2c has been recently identified in vibriosis cases from Atlantic cod. However, it was shown that the O2c sub-serotype might not belong to the O2 serotype, but in fact belongs to another serotype. To protect Atlantic cod against all the V. anguillarum serotypes (and subserotypes) which they are susceptible to, it is recommended that isolates from serotypes O1, O2a, O2b, O2c and O2d should all be included in a bacterin vaccine for cod. In order to determine which isolates from each of the serotypes to include in the vaccine, a variety of virulence factors of V. anguillarum were investigated in vitro. The interaction of some candidate isolates from O1, O2a and O2b serotypes (O2c and O2d were not identified at the time this part of the study took place) with cod phagocytic cells were studied using flow cytometry. Phagocytosis and respiratory burst of cod macrophages and neutrophils as well as cod serum killing of V. anguillarum were quantified. It was found that isolates within the same serotype displayed varying degrees of resistance to phagocytosis and the subsequent respiratory burst activity as well as that all the V. anguillarum strains tested were resistant to Atlantic cod serum killing. These in vitro assays were found to be very useful in assessing the virulence of V. anguillarum. The isolate within each serotype eliciting the highest percentage of positive phagocytic cells was selected in order to increase the antigen presentation pathway, thus theoretically enhancing the protection elicited by the vaccine. A multivalent formalin-inactivated non-adjuvanted vaccine was prepared which included all the serotypes previously described and was injected intraperitoneally into Atlantic cod. A bath challenge was performed on vaccinated and mock-vaccinated fish, 6 weeks post immunisation, using V. anguillarum isolates from the serotypes O2b, O2c and O2d that were not included in the vaccine. An excellent level of protection was obtained against O2b and O2d (relative percentage survival 100% and 96.4%, respectively), but the challenge with the sub-serotype O2c isolate did not produce any mortality in the control group and needs to be repeated. The vaccine formulation was very efficient at protecting Atlantic cod against vibriosis but further challenges need to be performed with other serotypes included in the vaccine (O1 and O2a), as well as with more isolates from the O2b, O2c and O2d sub-serotype. To conclude, Atlantic cod is a species which will certainly have a major influence in marine aquaculture, but many areas have to be improved. The development of an effective and broad range vaccine to protect cod against Vibrio anguillarum offers another advance which should help Atlantic cod aquaculture to reach its full potential.

Aphanomyces invadans is the pathogen that causes epizootic ulcerative syndrome (EUS), an economically devastating fish disease in southern Asia. The present thesis considered possible improvements to current methods of monitoring EUS, and examined the mechanisms of the host immune response to A. invadans in order to establish whether they could be enhanced to reduce the impact of EUS on aquaculture. Monoclonal antibody (MAb) technology was considered as a possible improvement to the histopathological methods currently used in diagnosis of EUS. Five MAbs were raised to day-old A. invadans germlings. Four gave weak reactions to A. invadans and cross-reacted with other Aphanomyces spp, though they may be useful for future studies on A. invadans. The other, designated MAb 3gJC9, only cross-reacted with the crayfish plague pathogen, A. astaci, and was used for the development of an immunohistochemistry protocol that may be of use in diagnosis. Immunohistochemistry with MAb 3gJC9, which recognised an extracellular product (ECP) of A. invadans, was specific to A. invadans in fish tissue, although it also recognised A. astaci in plague-infected crayfish. It also recognised the mycelium in fish infected with ulcerative mycosis, indicating that ulcerative mycosis is synonymous with EUS. Preliminary observations indicated that both ECPs and what appeared to be a hitherto unreported early stage of the mycelium are important in the pathology of EUS. Studies in vitro on the macrophages of EUS-susceptible giant gourami Osphronemus gouramy and silver barb Barbodes gonionotus, and EUS-resistant Nile tilapia Oreochromis niloticus, found that their macrophages were able to inhibit the growth of A. invadans. The macrophages of striped snakehead Channa striata did not inhibit A. invadans, which may account for their high EUS-susceptibility, especially as A. invadans strongly inhibited the respiratory burst of snakehead macrophages. Studies on humoral immune responses revealed that complement inhibited A. invadans in the case of snakeheads, gourami and barbs but not tilapia or swamp eels Monopterus albus. The humoral responses of the latter were very different to the four other species, and not elucidated. Low levels of anti A. invadans antibodies were found in tilapia and gourami from an EUS-endemic region, and high levels in snakehead. Snakehead antibodies appeared to be able to inhibit A. invadans even when complement was removed, but lower levels were produced at the low temperatures typically associated with EUS. A range of potential immunostimulants were screened for the ability to enhance resistance to EUS. The two successful products were administered as feed supplements to snakeheads and barbs that were subsequently injected intramuscularly with A. invadans. One, the algal extract Ergosan, showed some beneficial effects on snakeheads although the challenge was inconclusive. The other, the vitamin supplement Salar-bec, accelerated the cellular immune response and reduced mortality in snakeheads and barbs, and enhanced antibody production in snakeheads. The antibody response of snakeheads was further studied by comparing the anti- A. invadans antibody level, inhibitory activity of sera in vitro and protective capacity of sera from EUS-naïve snakeheads to that of snakeheads recently exposed to EUS and those subject to long term EUS-exposure. Sera of populations recently exposed to EUS showed an increased level of antibodies, but little improvement in inhibitory or protective activity. Sera from snakeheads that had endured long term exposure showed a wide range of antibody levels, but marked increases in inhibitory and protective activity. Antibodies cross-reacted with non-pathogenic Aphanomyces spp. in all cases.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 5 uppsatser.

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-11-09T16:36:26Z No. of bitstreams: 1 carlos-humberto-marques.pdf: 2761227 bytes, checksum: cf168e11c904e07038251644b2018901 (MD5) / Made available in DSpace on 2012-11-09T16:36:26Z (GMT). No. of bitstreams: 1 carlos-humberto-marques.pdf: 2761227 bytes, checksum: cf168e11c904e07038251644b2018901 (MD5) Previous issue date: 2005-05 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / Os anticorpos monoclonais possuem diversasaplicações em transplantes, na composição de conjuntosde reativos para diagnóstico, grande variedade de doenças auto-imunes e, principalmente, na terapia do câncer. A tecnologia de produção de anticorpos monoclonais recombinantes revolucionou a geração de imunoglobulinas, possibilitando a obtenção de anticorpos humanizados dirigidos a uma grande variedade de antígenos específicos. A baixa seletividade das metodologias atuais para diagnóstico e terapia de neoplasiasconstitui um dos principais empecilhospara a prática oncológica. Nesse particular, a utilização de imunoglobulinas submetidas à engenharia genética já é uma realidade e significa um avanço estratégico, abrangendo cerca de 25% do mercado biofarmacêutico global de proteínas terapêuticas. Este trabalho aponta os aspectos fundamentais à concretização da metodologia de humanização de anticorpos por transplante das regiões determinantes de complementaridade – CDR, com ênfase em uma proposta de produção do anticorpo anti – CD20 contrao Linfoma Não-Hodgkin. A introdução do Instituto de Tecnologia emImunobiológicos - Bio-Manguinhos nesse promissor e importante mercado de biofármacos através da implantação da metodologia de humanização do anticorpo monoclonal murino anti-CD20 é objeto desta dissertação. Viabilizar sua produção torna-se extremamente importante, tanto para a identificação precisa e precoce da enfermidade, quanto para atender um segmento do mercado brasileiro ainda desprovido de tratamento abrangente e eficaz. A apresentação do estudo dos anticorpos, sua estrutura e características, o estudo dosdiferentes sistemas de expressão, cultivo, purificação,bem como a proposta de reestruturação e redimensionamento do Laboratório de Tecnologia de Anticorpos Monoclonais, parcerias, colaborações, recursos humanos necessários e aspectos de mercado, são aqui considerados. / Monoclonal antibodies (Mabs) have several applications in transplants, reagents for diagnosis, a great variety ofauto-immune diseases and mainly, in cancer therapy. Mabs production employing recombinant echnologymade a revolution in immunoglobulinsgeneration, enabling the production of humanized antibodiesthat recognize specific antigens. The low selectivity of the current techniquesfor neoplasm diagnosis and therapy is one of the major impediments for oncology practice. In this regard, the use of eneticallyengineered immunoglobulins has become a reality and meansa strategic development comprising around 25% of the global biopharmaceutical market. This work shows the most important aspects in Mabs humanization through complementary determining regions (CDR) graft methodology, emphasizing a proposal ofanti-CD20 Mab production against non-Hodgkin lymphoma. Introducing the Instituto de Tecnologia emImunobiológicos – Bio-Manguinhos in this important and promising biopharmaceutical market through the establishment of humanization methodology is the main object of this dissertation. Making humanized Mabs production feasible is veryimportant not only for the earlyand precise identification of illnesses, but also to meet a demand of the Brazilian market that still lacks comprehensive and efficient treatment. The study of Mabs, their structureand properties, expression systems, cultivation, purification, new dimensions and structure of the laboratory, partnerships, cooperation,human resources and market analysis are considered herein.

Dissertation (PhD)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Introduction Despite advances in insulin therapy, the side effects associated with diabetes mellitus still remain. Pancreas transplantation has benefited diabetics with end-stage renal failure by reversing the diabetic state and preventing or reversing the progression of diabetes associated diseases. Currently the side effects associated with lifelong immunosuppression preclude pancreas transplantation as a viable treatment option for both type I and II diabetics. In the laboratory, transplanted rat foetal pancreata have been shown to be able to reverse the clinical signs of streptozotocin-induced diabetes in an isogeneic model. Reversal of diabetes by allogeneic foetal rat pancreas transplantation, although possible has proved to be more difficult due to fierce rejection of the grafts and the diabetogenic effects of conventional immunosuppressants. Aims One of the goals, focus and intentions of this laboratory study in rodents, is to contribute new information to the scientific literature. The potential to “reverse” the diabetic state by allogeneic foetal pancreatic transplantation, was the main stimulus for this study. Methods Foetal pancreata of 16-18 days gestation were transplanted into a surgically prepared renal subcapsular space. Immunosuppressive protocols used to prevent rejection of the allogeneic foetal rat pancreata included donor specific transfusion (DST), cyclosporine [a calcineurin inhibitor (CsA)], mycophenolate mofetil [a purine syntase inhibitor (MMF)], and a mouse anti-rat CD4 monoclonal antibody (W3/25). Immunosuppressants were used as monotherapies and in combination. Results Isogeneic foetal rat pancreas transplantation resulted in the growth and development of mature insulin producing islets of Langerhans at the site of engraftment. Allogeneic foetal pancreatic transplantation without immunosuppression resulted in complete rejection of the grafts at 14 days post-transplantation. Histological assessment of allografts at 14 and 30 days post-transplantation showed that CsA was able to prevent acute rejection in our rat models although graft scores and survival were improved if CsA was combined with MMF. Intraperitoneal anti-CD4 monoclonal injections were well tolerated, and if given daily effectively prolonged graft survival up to 30 days. Combining DST with anti-CD4 and CsA induction therapy provided long-term graft survival without daily immunosuppression. This combination, together with allogeneic foetal rat pancreas transplantation, was effective in reversing the clinical signs of experimentally induced diabetes. To my knowledge these are the first published results in which reversal of streptozotocin induced diabetes was achieved by fully MHC mismatched foetal rat pancreatic transplantation. Conclusion Foetal rat pancreatic transplantation is a potential source of endocrine replacement, which, with effective immunosuppression allows for the development of functional islets able to reverse the clinical signs of experimentally induced diabetes in an allogeneic rat model. An unique immunosuppressive protocol, with potential clinical relevance in the human, combines anti-CD4 mAb, CsA and DST induction therapy, which alleviates the burden of daily immunosuppression and associated side effects. / AFRIKAANSE OPSOMMING: Inleiding Ten spyte van die vordering met modeme insulienterapie bly die newe-effekte, waarmee diabetes mellitus geassosieer is, steeds ‘n probleem vir diabete. Diabetiese pasiente met eindstadium nierversaking trek geweldig voordeel uit nier-pankreasoorplantings wat die diabetes omkeer en die progressie van diabetesverwantesiektes voorkom of selfs omkeer. Die newe-effekte van lewenslange immuunonderdrukking skakel pankreasoorplanting uit as ‘n lewensvatbare behandelingsopsie vir tipe I of II diabete. In ‘n streptozotosien-gei'nduseerde diabetiese rotmodel kan isogenei'ese fetale pankreasoorplanting die kliniese tekens van diabetes omkeer. Die omkering van streptozotosien-gei'nduseerde diabetes deur allogeneiese fetale pankreasoorplanting behoort moontlik te wees indien verwerping en die diabetogeniese newe-effekte van konvensionele immuunonderdrukkers oorkom word. Doelstellings Een van die mikpunte, fokusse en oogmerke van hierdie laboratorium studie in knaagdiere, is om ‘n betekenisvolle bydrae tot nuwe kennis in die wetenskaplike literatuur, te maak. Die potensiaal om die diabetiese toestand deur allogeneiese fetale pankeasoorplanting om te keer, was die hoof stimulus vir die studie. Metodes Fetale rotpankreata van 16-18 dae gestasie was in ‘n chirurgies voorbereide spasie onder die nierkapsel oorgeplant. Immuunonderdrukkende protokolle, vir die voorkomming van verwerping van die allogeneiese fetale pankreasoorplantings, het donorspesifiekeoortappings (DST), siklosporien [‘n kalsineurien inhibitor (CsA)], mikofenolaat mofetiel [‘n purien sintase inhibitor (MMF)] en ‘n anti-rot CD4 monoklonale antiliggaam (W3/25) ingesluit. Die immuunonderdrukkers is as mono- of as kombinasieterapie gebruik. Resultate IsogeneTese fetale rotpankreasoorplanting het tot die ontwikkeling van volwasse insulienproduseerende eilande van Langerhans gelei, wat die kliniese tekens van streptozotosien-gei'nduseerde diabetes kon omkeer. Allogenei'ese fetale rotpankreasoorplanting sonder immuunonderdrukking het tot algehele verwerping van die oorplanting binnel4 dae na oorplanting gelei. Histologiese beoordeling van die oorplantings 14 en 30 dae na oorplanting het getoon dat CsA akute verwerping van fetale pankreasoorplantings in die rotmodelle voorkom. Indien CsA met MMF gekombineer word, word die oorplantings-telling en oorlewing verbeter. Intraperitoneale anti-CD4 monoklonale inspuitings was goed verdra, en indien daagliks toegedien, het dit die oorlewing van die pankreasoorplantings effektief tot 30 dae verleng. Die kombinasie van DST, anti-CD4 en CsA induksieterapie het tot langtermyn oorlewing van die pankreasoorplantings gelei sonder verdere daaglikse immuunonderdrukking. Die induksieterapie in kombinasie met allogenei'ese fetale pankreasoorplanting was effektief in die omkering van die kliniese tekens van streptozotosien-gei'nduseerde diabetes in die rot. Hierdie is, sover ek weet, die eerste keer dat omkering van streptozotosien-gei'nduseerde diabetes suksesvol met ‘n volledige MHC onverenigbare allogenei'ese fetale pankreasoorplanting behaal is. Gevolgtrekkings Fetale rotpankreasoorplanting is ‘n potensiele bron vir endokrien vervangingsterapie, wat met effektiewe immuunonderdrukking tot die ontwikkeling van funksionele eilande van Langerhans lei, wat die vermoe het om die kliniese tekens van experimenteel-ge'induseerde diabetes in ‘n allogeneiese rotmodel om te keer. ‘n Unieke immuunonderdrukkingsprotokol, met kliniese relevansie, kombineer DST met anti-CD4 mAb en CsA induksieterapie wat die las van daaglikse immuunonderdrukking en die geassosieerde newe-effekte van konvensionele immuunonderdrukking verlig.

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Almost a century has elapsed since the antigen-antibody interaction was first recognised as the basis of an immune response (Ehrlich, 1897). However, it was only in the 1930s, with the development of improved technologies that this concept was better understood, and led to the discovery of the amazing diversity and specificity of antibody molecules (Landsteiner, 1933). Theoretically, it is possible to make antibodies to a variety of biological substances and other chemicals, and therefore they are ideally suited as specific recognition elements to be used for analytical, cytological, functional, therapeutic and biochemical purposes. The development of the radioimmunoassay (RIA) thirty five years ago, revolutionised research in many areas of clinical and scientific investigation. This technique evolved rapidly from the discovery made by Berson et al. in 1956 that antibodies to insulin could be detected in patients treated with this hormone, by measuring the binding of radiolabeled insulin to these antibodies. Although in the past RIAs have been the most important assay system employing antibody and labelled tracer, the limitation was that reliance had to be placed on the chance development of a good polyclonal antibody. These shortcomings stimulated the search for monospecific antibodies of reproducible quality and sufficient quantity. The development and introduction of monoclonal antibody technology brought about a revolution in immune serology (Kohler and Milstein, 1975). Establishment of immortal cell lines which contained the genetic elements of antibody-producing cells was achieved by fusion between a myeloma cell line and spleen cells from an immunised donor. The resulting hybrids had the essential properties of both parents, namely, permanent growth and a high capacity for the synthesis and secretion of immunoglobulins, normally characteristics of plasmacytomas, together with the genetic elements defining a specific antibody. Gestational trophoblastic disease (GTD) is a neoplastic condition of the trophoblast and occurs as molar pregnancy in a benign or invasive form, or as choriocarcinoma in a malignant form. Effective therapy has been developed for the treatment of both choriocarcinoma and molar pregnancy, but the key to successful management of these patients lies in their prompt diagnosis and careful monitoring of response to treatment (Green-Thompson, 1986). Fortuitously, these tumours elaborate the human chorionic gonadotrophin hormone (hCG) and its free alpha (a) and beta (B) subunits and hence a ready marker for the tumour exists. Human chorionic gonadotrophin is one of a group of glycoprotein hormones, which includes luteinising hormone (LH), follicle stimulating hormone (FSH) and thyroid stimulating hormone (TSH). These hormones are composed of two dissimilar subunits designated a and B, which are bound non-covalently in the intact molecule. The B-subunit of each glycoprotein hormone is unique and is responsible for the respective biological and immunological properties of the glycoproteins. In contrast, all four hormones possess an identical a-subunit which is coded for by a single gene (Fiddes and Goodman, 1979). The measurement of hCG and its free B-subunit, as so-called BhCG, for the diagnosis and monitoring of therapy in patients with GTD is now routinely practised throughout the world (Vaitukaitis et al., 1972). However it has been demonstrated by Bagshawe (1975) that when serum BhCG can no longer be measured by current RIA methods, up to 10" tumour cells may remain undetected. In addition, there have been isolated reports of two patients with choriocarcinoma in whom BhCG was undetectable in the serum but who appeared to be secreting only the a-subunit (Dawood et al, 1977). Furthermore, it has been suggested that measurement of free a-subunit rather than intact hCG or the free B-subunit is a more effective means of detecting persistent trophoblastic disease as well as tumour recurrence following treatment (Quigley et al, 1980a and b). Radioimmunoassays which measure the free a-subunit of hCG have been developed, but in general lack the specificity and sensitivity required (Gaspard et al, 1980; Kohorn et al, 1981). These assays employ polyclonal antisera which also detect epitopes common to the pituitary gonadotrophins. Thus there is a need to produce monoclonal antibodies which recognise regions of the free a-subunit which are hidden in the intact gonadotrophins. Such antibodies would provide the required specificity for use in RIAs but are limited in their use by their inherent lack of high affinity for the antigen. Fortunately, this drawback may be overcome by using monoclonal antibodies as labelled reagents in an alternative assay system, the immunoradiometric assay (IRMA), described by Miles and Hales (1968). The IRMA, particularly the two-site sandwich version of the assay, has been shown to provide greater sensitivity in addition to allowing enhanced specificity. This is a consequence of the use of two antibodies in excess to detect the analyte, each directed at a different epitope on the target molecule. The first antibody, referred to as the capture antibody, is usually linked to a solid-phase to facilitate easy separation and is added in excess relative to the target hormone to enhance antibody-antigen interaction, thereby allowing increased sensitivity in the measurement of analyte. The second antibody, referred to as the detection antibody, is labelled with a radioactive isotope or an enzyme to detect antigen already bound to the capture antibody. The application of monoclonal antibodies specific for the free a-subunit to a highly sensitive IRMA format is an obvious need. Hence this study was undertaken firstly, to raise and characterise monoclonal antibodies to the free a-subunit, secondly to develop an IRMA using these antibodies and finally to establish whether measurement of free a-subunit has any clinical advantage. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1990.