To determine the antigenic relatedness of the different developmental stages of Cryptosporidium parvum, monoclonal IgG3 antibody (mAb), Cmg-3, was produced by immunizing mice with partially purified merozoites. The monoclonal Cmg-3 reacted with a 3.5 kDa antigen of sporozoites in western blots and appeared to react with cell surface antigens of air-dried merozoites and sporozoites using immunofluorescence (IF). Additional mAbs, C6B6 (IgG1) and C4A1 (IgM), which react with a 20 kDa and multiple sporozoite antigens, respectively, were employed for immunoelectron microscopic studies with Cmg-3. These mAbs showed similar (surface/cytoplasmic) immunoelectron microscopic colloidal gold labeling patterns with all C. parvum life cycle stages. The three mAbs were also examined for potential modulation of cryptosporidial infections in vivo by daily oral mAb administration to oocyst-inoculated neonatal mice. Monoclonal-treated neonatal mice were sacrificed four and eight days post infection (pi). Differences in infection rates were observed among the treatment groups (p < .05). Suckling mice treated daily with orally administered mixtures of mAbs (ascitic fluids) showed significantly reduced parasite loads compared to control mice at four and eight days pi, while suckling mice receiving mAb Cmg-3 alone showed significant differences only at four days pi. Passive transfer of immunity using hyperimmune bovine colostrum was performed to determine the therapeutic and prophylactic efficacy of daily oral administration of anti-C. parvum antibody on the manifestation of cryptosporidial disease in neonatal mice as a model for treating cryptosporidiosis in immunocompromised patients. Hyperimmune colostrum was found to provide therapeutic and prophylactic efficacy against cryptosporidiosis in neonatal mice. Significantly fewer (p < 0.05) stages of C. parvum were found in mice that received hyperimmune skim colostrum (HSC) or hyperimmune original colostrum (HC) than in those treated with control colostrum (CC) or saline. Using IF, antigen-specific IgG in HSC and HC to C. parvum was 35 times greater than that of CC. There was no significant difference between groups treated with HSC or HC (p < .05), which suggests that the immunoglobulins, other biologically active factors such as cytokines, or both, might be active factors of immunity against cryptosporidiosis.

Monoclonal antibodies (mAbs) have been essential tools in the elucidation of the immune system of mammals, and their application to identify surface molecules on leukocytes have allowed important functions of these cell to be identified (such as receptors that bind antigens, ligands involved in cell to cell signaling or in initiating immune response activity). Not only have mAbs been used to discriminate cells during different stages of cell development, but have also assisted in understanding the dynamics of molecules expressed during functional processes. Such molecules detected on human leukocytes are called human leukocyte differentiation antigens or HLDA. In order to group the antibodies that detect similar molecules and have similar patterns of reaction, immunologists have organised the mAbs that bind to these antigens into Clusters of Differentiation (CD). So far, there are about 350 leukocyte surface molecules detected by mAbs with a CD nomenclature for human leukocytes (www.hcdm.org). In fish immunology there is a great need to produce mAbs that are able to differentiate the various components of the fish immune system to assist in the elucidation of the fish immune system. The present study was an endeavour to develop and characterise mAbs that could be accredited to such scheme. A better understanding of the fish immune system is urgently required so that effective strategies of control can be developed for significant diseases during fish farming. Monoclonal antibodies were prepared by immunizing mice with thymic leukocytes from rainbow trout. The leukocytes were activated with the lectin Concanavalin A to promote the activation and proliferation of the target T-cell population. The selection of clones producing antibodies during screening was performed on the basis of the response of the supernatant from hybridomas using three consecutive assays. First, selection was determined by the positive staining of cells from the thymus in a Dot blot assay. Secondary screening was performed by means of flow cytometry (FCM) and the criterion for selection was the preferential detection of leukocytes gated in the lymphocyte region. Finally, the positive supernatants from hybridomes were evaluated to determine their effectiveness in the detection of modifications in the labelled cells during a multiple way activation by detection of foreign histocompatibility complex enhanced with mitogens. Monoclonal antibody TcOm15 was selected from 564 hybridomas produced and then used to stain cells from various Rainbow Trout tissues. It was clear from FCM, microscopy and Western blot analysis that mAb TcOm15 not only reacted with thymic cells but also with cells from other tissues. Differential staining of cells with mAb TcOm15 was observed with 27.1 ±1.4 % of leukocytes from peripheral blood leukocytes (PBL) stained in comparison to 2.0 ±0.2 % from the thymus, 13.8 ±0.4 % from the spleen, and 5.6 ±0.6 % cells stained from head kidney. The labeled cells showed characteristics of lymphocytes and monocytes, presenting a distinctive staining in immunohistochemistry and confocal microscopy. Western blot analysis, using electrophoresed proteins under denaturing conditions with leukocytes from several different tissues, showed that mAb TcOm15 did not detect a single protein. At least three proteins appeared to be identified by the mAb at 105, 160 and 200 kDa. The proteins were identified as α Actinin-4, non-erythroid Spectrin αII chain or Ig-like protein and non-muscle Myosin (MYH10) by MALDI-TOF analysis. Three of these identities are for compositional molecules for the cytoskeleton of different types of cells, and one it is associated to immunoglobulin superfamily. The identification of these proteins by mAb TcOm15 suggests an ability of this mAb to detect a specific function, possibly related with the synchronicity of expression or interaction of cytoskeleton-membrane proteins forming a multiprotein complex. Another possibility is as a carrier role for a protein during interactions. Colocalization of the mAb with F actin from the cytoskeleton was also observed suggesting the possibility that mAb TcOm15 detects a specific site in a multi-protein complex from the cytoskeleton. The molecule detected showed down-regulation in a dose dependant way with Concanavalin A and the expression was almost lost following stimulation of cells with phorbol 12-myristate 13-acetate stimulation. Leukocytes from the PBL and thymus up-regulated the expression of the TcOm15 molecule under mitogenic conditions in vitro, and results from in vivo experiments suggested the possibility of up-regulation on thymic cells. In conclusion, the results obtained in the present study provide information on a potentially useful marker (mAb TcOm15) for a cytoskeleton-membrane antigen that is modulated during stimulation of teleost lymphocytes. Additionally, this may enable insights into the relationship between cytoskeletal proteins and membrane associated immunoglobulin. Future research is necessary in order to explain this relationship and to determine the functional participation of the TcOm15 molecule during the activation of rainbow trout cells.

As modificações das lipoproteínas de baixa densidade (LDL) são uma etapa essencial na aterogênese pois acarretam a geração de LDL eletronegativa [LDL(-)] que apresenta propriedades quimiotática, citotóxica, imunogênica e pró-inflamatória. O objetivo deste trabalho foi a produção de hibridomas secretores de anticorpos monoclonais anti-LDL(-), a clonagem dos genes que codificam para as cadeias variáveis destes anticorpos, e sua expressão como fragmentos de anticorpo de cadeia única (scFv). A LDL(-) isolada de plasma humano foi utilizada como antígeno para imunização de camundongos BALB/c. Após triagem dos clones, dois anticorpos monoclonais foram obtidos baseados em sua reatividade pela LDL( -) e não pela LDL nativa: 1A3H2 (1A3) e 2C7D5F10 (2C7). Os cDNAs codificante para a cadeia pesada (VH) e cadeia leve (VL), de ambos os anticorpos, foram obtidos por meio de RT-PCR utilizando bibliotecas de oligonucleotídeos que reconhecem todas os genes de domínios variáveis das famílias de VH e VL murinas. Os genes da VH e VL obtidos foram clonados no vetor pGEM-T Easy (Promega&#174;) e suas seqüências determinadas. A VH do anti-LDL(-) 1A3 pertence família J558.84 e fragmento gênico JH2, enquanto sua VL pertence a família 8.24 e fragmento gênico Jk5. A VH do anti¬-LDL(-) 2C7 pertence a família Vmu 3.2 (J558) e fragmento gênico JH4, enquanto sua VL pertence a família 8.24 e fragmento gênico Jk5. A partir disso, oligonucleotídeos sintéticos foram sintetizados a fim de clonar estes segmentos gênicos no vetor pPlgLE de expressão em Pichia pastoris. Foram realizadas três construções: o scFv 1A3, scFV 2C7 e um scFv híbrido (VH do 1A3 e VL do 2C7). Das três construções obtidas, conseguimos expressar o scFv do anti-LDL 2C7D5F10 que demonstrou ser capaz de reconhecer o antígeno. A proteína recombinante expressa tem grande potencial de ser usada no diagnóstico clínico incluindo imunoensaios in vitro e como reagentes para exames que envolvam a obtenção e análise de imagens. / Oxidative modification of low-density lipoproteins (LDL) is an essential step in atherogenesis, generating electronegative LDL [LDL(-)], which has chemotactic cytotoxic, immunogenic and proinflammatory properties. The aim of this study was the generation of anti-LDL(-) mAbs, the cloning of the genes that code for their variable domains and their expression as single-chain Fv (scFv). LDL(-) was isolated from human blood plasma and used as an antigen for immunization of Balb/c mice. Upon screening, two different mAbs were selected based on their ability to recognize LDL(-) and not native LDL: 1A3H2 (1A3) e 2C705F10 (2C7). The cDNAs that code for VH and VL were obtained by RT-PCR using specific immunoglobulin primer libraries wich recognize all VH and VL murine families. The VH and VL genes were cloned in pGEM-T Easy (Promega&#174;) and sequenced. The anti-LDL(-) 1A3 uses a VH segment from J558.84 and a JH2 segment, while VL uses a 8.24/Jk5 segments. The anti-LDL(-) 2C7 uses a VH segment from Vmu 3.2 (J558) and a JH4 segment, while VL uses a 8.24/Jk5 segments. Oligonucleotides were synthetized and those gene segments were cloned in pPIGLE a Pichia pastoris immunoglobulin expression vector. We obtained three scFv constructions: scFV 1A3, scFv 2C7 and a husk hybrid, harboring 1A3 VH and 2C7 VL. Among those, we expressed the scFv anti¬-LDL(-) 2C7 that are able to recognize the antigen. The recombinant protein has a great potential for clinicai diagnostic applications, including in vitro immunoassays and as imaging reagents.

Evidências epidemiológica e imunológica sugerem que o desenvolvimento da imunidade natural contra doença meningocócica pode está associado com a reação cruzada de antígenos em comuns com Neisseria meningitidis e outras bactérias comensais, como Neisseria lactamica. O Objetivo deste trabalho foi de investigar a imunogenicidade de antígenos de vesículas de membrana externa (OMV) de N. lactamica, com ou sem a presença de Bordetella pertussis (BP), utilizada como adjuvante. Grupos de camundongos neonatos da linhagem BALB/c foram imunizados com antígenos de N. lactamica. Os resultados de nossos estudos mostraram o predomínio de altos títulos de anticorpos dos isótipos IgG e IgM com alta e intermediária avidez, depois das imunizações pela via (i.n) com N. lactamica. A análise do soro por immunoblot mostrou proteínas com reatividade cruzada entre as espécies do gênero Neisseria e os anticorpos monoclonais utilizados neste trabalho. Estes resultados sugerem que antígenos de N. lactamica e N. meningititdis em comum, possam ser importantes na imunidade natural contra doença meningocócica, e no desenvolvimento de vacina. / Immunological and epidemiological evidences suggest that the development of natural immunity to meningogoccal disease may be associated with crossreactive antigens together with Neisseria meningitidis and other commensal bacteria, like Neisseria lactamica. The present study aimed to investigate the immunogenicity of antigens of outer membrane vesicles (OMV) of N. lactamica with or without the presence of Bordetella pertussis (BP) used as an adjuvant. Groups of neonate BALB/c mice were immunized intranasally antigens of N. lactamica. The results of our studies showed the predominance of high titers of antibodies of IgG and IgM isotipes with high and intermediate avidity after intranasal immunization with N. lactamica. Immunoblot analysis of serum showed cross-reactivity proteins between the species of the gender Neisseria and the monoclonal antibodies used in this study. These results suggest that antigens of N. lactamica and N. meningitidis in common may be important in natural immunity against meningogoccal disease and in the development of vaccine.

No description available.

The aim of this thesis was to establish the therapeutic value of the IFITM1 monoclonal antibodies and to design and develop therapeutically valuable recombinant monoclonal antibodies so as to study the implication of these novel antibodies in cancer therapy. Cancer metastasis is one of the main interests that has given rise to the design and development of innovative strategies for cancer therapeutics. The Interferon Induced Transmembrane Protein 1(IFITM1), a notable member of the IFITM family of proteins has been identified as one of the most up-regulated trans-membrane proteins in metastatic breast cancer and cervical adenocarcinoma. This interferon-regulated protein is also involved in cell migration, invasion in glioma and squamous cancers. This PhD aimed to study IFITM1 as a pro-invasive cancer target by the use of IFITM1 monoclonal antibodies that were raised against the extracellular domain of the human IFITM1 gene. The epitope mapping of IFITM1 revealed the binding activity of the IFITM1 monoclonal antibody. This gave the opportunity to design and generate to new IFITM1-specific molecular tools, in the form of recombinant IFITM1 targeted murine scFv antibody, IFITM1-CPG2 yeast fusion protein antibody for potential application in ADEPT as well as a Mouse-Human Chimeric IFITM1 antibody secreting mammalian cell line. The immunohistochemical staining of IFITM1 in tissue micro array from breast, colon and oeosphegal cancer has revealed that the majority of these cancers produce this protein. However, IFITM1 is over produced in cervical cancer indicating it’s selective over expression in cervical cells. This PhD endeavored to investigate the expression of IFITM1 at a translational and transcriptional level and to study the clinical significance of IFITM1 in cervical cancer. The antibody dependent cell mediated cytotoxic activity of the chimeric IFITM1 antibody was found to be cytotoxic to SiHa cells in vitro. In the future these molecular tools could be used to regulate and further characterize the activity of this transmembrane protein antibody. In an effort to better understand the mechanisms that regulate the activity and the over production of the IFITM1 gene and its interacting proteins, a proteomic screen of cervical cancer cells was carried out using data-independent SWATH-MS on an AB SCIEX TripleTOF™ mass spectrometer. This Mass Spec analysis provided us with a host of IFITM1 biomarkers and revealed that the IFITM1 gene and its binding proteins also cross link with the IRF1 pathway. The data presented in this thesis, demonstrates that the IFITM1 gene can be targeted to either stimulate or inhibit IFITIM1 signaling to engage IFITM1 as a potential pro-invasive extracellular receptor as a target in antibody cancer therapy. In summary, this thesis aimed to confirm the activity and the binding specificity of the IFITM1 antibody. Additionally, this thesis demonstrated a promising application of the recombinant antibody in the ADEPT technology. Characterization of IFITM1 mAb effector functions indicated that the antibody was cytotoxic to cervical cancer cells. This highlights an important element in the immune suppressive tumour microenvironment. And finally, this thesis also provides the basis for the production of recombinant mouse human chimeric antibodies that are a part of a new group of immunotherapeutic molecules paving the way for cancer therapeutics.

Orientador: Elenice Deffune / Coorientador: Márjorie de Assis Golim / Banca: Rosimeire Aparecida Roela / Banca: Paulo Inácio da Costa / Banca: Rosimeire Aparecida Roela / Resumo: O sistema imunológico é dividido em imunidade natural e adquirido (humoral e celular). Os linfócitos B são os principais efetores da resposta humoral. Junto aos linfócitos T, mediam diversas reações imunológicas. Todos os leucócitos possuem antígenos de superfície (clusters of differentiation - CD) determinados que possuem as mais diferentes funções. As expressões destes CDs podem variar na maturação e na presença de patologias, sendo as de maior prevalência e gravidade as leucemias e linfomas, tornando-se marcadores importantes que podem ser avaliados por citometria de fluxo ou imunoquímica através do uso de anticorpos monoclonais murinos (AcMm). Após produção dos AcMm é necessário caracterizar e validá-los. Utilizou-se 11 clones que apresentaram especificidade somente contra linfócitos B. Pela técnica de Western Blotting, 5 anticorpos (3 do tipo IgM e 2 do tipo IgG) foram escolhidos de acordo com sua possível especificidade e importância clínica. A validação dos anticorpos tipo IgM foi realizada por citometria de fluxo utilizando anticorpo comercial para comparação de quantidade de células marcadas, sendo testados em 20 amostras de indivíduos normais e 20 de indivíduos portadores de neoplasias hematológicas diversas. O LINB B, que foi comparado com o anti-CD171 e anti-CD45RA, apresentando diferença estatística somente em relação ao anti-CD45RA, e identidade com o anti-CD171. O LINB C, que foi comparado com o anti-CD20 e anti-CD19, não apresentou diferença estatística significante quando frente a ambos anticorpos comerciais. No teste de regressão linear, houve maior correlação dos resultados com o anti-CD19. O LINB E foi comparado somente contra o anti-CD107b, havendo grande identidade entre os dois. Dos resultados apresentados, conclui-se que o LINBs B e E apresentam grandes chances de serem específicos contra... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The immunological system is divided into: natural immunity and acquired immunity (celular and humoral responses). The B lymphocytes are the main effectors of the humoral response. Together with the T lymphocytes, they make several immunological reactions. All leucocytes have antigens on the surface (clusters of differentiation - CD) that possess lot of functions. The expressions of these CDs may be altered during maturation and pathologies, like leukemia and lymphomas, becoming important markers that can be evaluated by flow cytometry or immunochemistry thought murine monoclonals antibodies (Mab). After production of Mabs it's necessary characterize and validated them. We used 11 clones that presented Mab against B lymphocytes only. By Western Blotting method 5 Mab (3 IgM and 2 IgG) were chosen according you possible especifity and clinical importance. The validation of IgM Mabs were made by flow cytometry using commercial antibody to compare the quantity of marked cells, being used 20 samples from normal people and 20 samples from person with hematological cancer. The LINB B, compared to anti-CD171 and anti-CD45RA, presented statistical difference from anti-CD45RA and identity to anti-CD171. The LINB C, compared to anti-CD19 and anti-CD20, didn't presented any statistical difference from both commercial antibodies, although it correlates better with anti-CD19. The LINB E was compared to anti-CD107b, where it appears great identity between then. By the present results, we conclude that LINB B and E need multicentre studies to expand validation, and LINB C, needs to increase the samples to have a statistical validation. The two IgGs LINB, possible anti-CD138, weren't test yet. / Mestre

Monoclonal antibodies (mAbs) represent one of the fastest growing classes of therapeutic proteins. This success is due to a number of attractive properties such as high binding affinity, specificity, low immunogenicity and high aqueous solubility. Despite this, mAbs can suffer from undesirable physical instabilities, especially reversible self-association (RSA), which can lead to aggregation and phase separation. One aspect of formulation is therefore to find solution conditions which minimise mAb aggregation propensity during storage at high concentrations. Hence, the buffer, excipient and pH must be carefully considered to obtain the optimal formulation. Currently, if a platform formulation process is non-ideal for a particular candidate mAb, then an alternative strategy is to utilise high-throughput screening to measure various physical parameters indicative of physical stability. Arginine (in the form of hydrochloride salt Arg·HCl) is often used in formulations exhibiting high RSA and a propensity for aggregation. The interaction of Arg with the protein surface is complex and dependent on both the salt form and concentration. Here the focus was on the glutamate salt of arginine (Arg·Glu), to quantify its effect on mAb conformational and colloidal stability under different pH conditions. Arg·Glu was able to decrease the propensity of the mAbs to aggregate, particularly at pH values closer to their pI.The work also included the use of in vitro cell culture models to examine cell viability in the presence of the various arginine salts over a range of osmolalities. Whilst Arg·Glu is composed of two naturally occurring amino acids and both of which are considered non-toxic individually, the effect of the increased concentrations of their combination, on cells has not been explored previously. In vitro cell lines were chosen to represent the subcutaneous tissue, the effect of Arg·Glu on cell viability was compared against NaCl, Arg·HCl and sodium glutamate (NaGlu). The work concluded there was no additional toxicity associated with the presence of Arg·Glu in the cell culture models studied, therefore Arg·Glu has the potential as an excipient as it reduces aggregation and is nontoxic. Another aspect of the work was to assess the use of solution NMR spectroscopy as an orthogonal technique in mAb formulation characterisation. 1H NMR spectroscopy was used to measure a number of experimental parameters for high concentration mAb solution. The work proposed that 1H NMR spectroscopy can serve as a valuable orthogonal method for mAb characterization and formulation.

As modificações das lipoproteínas de baixa densidade (LDL) são uma etapa essencial na aterogênese pois acarretam a geração de LDL eletronegativa [LDL(-)] que apresenta propriedades quimiotática, citotóxica, imunogênica e pró-inflamatória. O objetivo deste trabalho foi a produção de hibridomas secretores de anticorpos monoclonais anti-LDL(-), a clonagem dos genes que codificam para as cadeias variáveis destes anticorpos, e sua expressão como fragmentos de anticorpo de cadeia única (scFv). A LDL(-) isolada de plasma humano foi utilizada como antígeno para imunização de camundongos BALB/c. Após triagem dos clones, dois anticorpos monoclonais foram obtidos baseados em sua reatividade pela LDL( -) e não pela LDL nativa: 1A3H2 (1A3) e 2C7D5F10 (2C7). Os cDNAs codificante para a cadeia pesada (VH) e cadeia leve (VL), de ambos os anticorpos, foram obtidos por meio de RT-PCR utilizando bibliotecas de oligonucleotídeos que reconhecem todas os genes de domínios variáveis das famílias de VH e VL murinas. Os genes da VH e VL obtidos foram clonados no vetor pGEM-T Easy (Promega&#174;) e suas seqüências determinadas. A VH do anti-LDL(-) 1A3 pertence família J558.84 e fragmento gênico JH2, enquanto sua VL pertence a família 8.24 e fragmento gênico Jk5. A VH do anti¬-LDL(-) 2C7 pertence a família Vmu 3.2 (J558) e fragmento gênico JH4, enquanto sua VL pertence a família 8.24 e fragmento gênico Jk5. A partir disso, oligonucleotídeos sintéticos foram sintetizados a fim de clonar estes segmentos gênicos no vetor pPlgLE de expressão em Pichia pastoris. Foram realizadas três construções: o scFv 1A3, scFV 2C7 e um scFv híbrido (VH do 1A3 e VL do 2C7). Das três construções obtidas, conseguimos expressar o scFv do anti-LDL 2C7D5F10 que demonstrou ser capaz de reconhecer o antígeno. A proteína recombinante expressa tem grande potencial de ser usada no diagnóstico clínico incluindo imunoensaios in vitro e como reagentes para exames que envolvam a obtenção e análise de imagens. / Oxidative modification of low-density lipoproteins (LDL) is an essential step in atherogenesis, generating electronegative LDL [LDL(-)], which has chemotactic cytotoxic, immunogenic and proinflammatory properties. The aim of this study was the generation of anti-LDL(-) mAbs, the cloning of the genes that code for their variable domains and their expression as single-chain Fv (scFv). LDL(-) was isolated from human blood plasma and used as an antigen for immunization of Balb/c mice. Upon screening, two different mAbs were selected based on their ability to recognize LDL(-) and not native LDL: 1A3H2 (1A3) e 2C705F10 (2C7). The cDNAs that code for VH and VL were obtained by RT-PCR using specific immunoglobulin primer libraries wich recognize all VH and VL murine families. The VH and VL genes were cloned in pGEM-T Easy (Promega&#174;) and sequenced. The anti-LDL(-) 1A3 uses a VH segment from J558.84 and a JH2 segment, while VL uses a 8.24/Jk5 segments. The anti-LDL(-) 2C7 uses a VH segment from Vmu 3.2 (J558) and a JH4 segment, while VL uses a 8.24/Jk5 segments. Oligonucleotides were synthetized and those gene segments were cloned in pPIGLE a Pichia pastoris immunoglobulin expression vector. We obtained three scFv constructions: scFV 1A3, scFv 2C7 and a husk hybrid, harboring 1A3 VH and 2C7 VL. Among those, we expressed the scFv anti¬-LDL(-) 2C7 that are able to recognize the antigen. The recombinant protein has a great potential for clinicai diagnostic applications, including in vitro immunoassays and as imaging reagents.

Monoclonal antibodies (MCA) produced against infectious laryngotracheitis virus (ILTV) of chickens reacted in western blotting experiments with several different ILTV protein bands in the absence of tunicamycin which inhibits carbohydrate synthesis. Most of the MCA lost their reactivity in western blotting experiments when extracts of tunicamycin-treated ILTV CELC were used, suggesting their specificity for carbohydrate-based epitopes. In an indirect immunofluorescence test most of the MCA bound primarily to cytoplasmic antigens except some MCA which bound primarily to nuclear antigens. Additivity ELISA was also performed to study whether MCA are against the same epitope or different epitopes. The polymerase chain reaction (PCR) was developed as a diagnostic technique for detection of ILTV using primers made from a portion of the ILTV thymidine kinase gene. The 647-basepair amplified ILTV PCR product was labeled to create a non-radioactive, biotinylated DNA probe. Hybridization was performed using the probe to detect ILTV. Both PCR and hybridization detected ILTV, and neither hybridization nor PCR gave positive results with any other pathogen. Hybridization was specific for ILTV, However, slight hybridization occurred with CELC DNA when relatively relaxed conditions were used. In another experiment, diagnostic tests to detect ILTV in tracheas of experimentally-infected chickens, including the indirect fluorescent antibody test (IFAT), immunoperoxidase (IP), virus isolation (VI), histopathology, PCR, and hybridization, were performed and compared. Using virus isolation as a reference, the sensitivity and specificity of the tests were calculated. The IP test and IFAT performed better than any other test used in this study. / Graduation date: 1995