Analysis of the serological variability of Lettuce mosaic virus using monoclonal antibodies and surface plasmon resonance technology

A panel of 19 monoclonal antibodies (mAbs) was used to study the immunological variability of Lettuce mosaic virus (LMV), a member of the genus Potyvirus, and to perform a first epitope characterization of this virus. Based on their specificity of recognition against a panel of 15 LMV isolates, the mAbs could be clustered in seven reactivity groups. Surface plasmon resonance analysis indicated the presence, on the LMV particles, of at least five independent recognition/ binding regions, correlating with the seven mAbs reactivity groups. The results demonstrate that LMV shows significant serological variability and shed light on the LMV epitope structure. The various mAbs should prove a new and efficient tool for LIVIV diagnostic and field epidemiology studies.

Taenia saginata: Production and characterization of monoclonal antibodies against Taenia saginata metacestode antigens

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

IMMUNOCYTOCHEMICAL DIFFERENTIATION OF REACTIVE HYPERPLASIA FROM FOLLICULAR LYMPHOMA USING MONOCLONAL-ANTIBODIES TO CELL-SURFACE AND PROLIFERATION-RELATED MARKERS

We tested the hypothesis that a panel of antibodies to cell surface, cytoplasmic, and nuclear antigens could reliably distinguish the cells composing reactive germinal centers from those composing follicular lymphoma. Immunocytochemistry was performed on deparaffinized sections of methacarn-fixed lymph node and tonsil (15 cases of reactive hyperplasia and 14 cases of follicular lymphoma) using antibodies to the nerve growth factor receptor (NGFR5), bcl-2 protein (124), proliferating cell nuclear antigen (PCNA; 19A2), and CD45RA (MT2). In 100% of cases of reactive hyperplasia, both MT2 and 124 showed positive immunostaining of mantle zone and scattered interfollicular lymphocytes, but in all cases there was a sharply demarcated absence of immunostaining of germinal center cells. However, diffuse immunostaining of follicular centers with MT2 (64%) and 124 (93%) and scattered intervening cells were seen in follicular lymphoma. The combination of antibodies to CD45RA and bcl-2 yielded positive immunostaining of follicular center cells in 93% of follicular lymphomas. The germinal center cells of reactive hyperplasia showed >75% nuclear positivity with antibodies to PCNA, in contrast to the follicular lymphoma cells, which showed variable PCNA indices ranging from 25 to >75%. A minority of follicular lymphoma cases (29%) showed PCNA indices comparable with those seen in cases of reactive hyperplasia. Antibodies to NGFR were positive in all cases of reactive hyperplasia and in 79% of cases of follicular hyperplasia, although the immunostaining intensity was generally decreased in follicular hyperplasia. In summary, antibodies to bcl-2 appear to be superior to those to CD45RA in distinguishing reactive hyperplasia from follicular lymphoma. Reactive hyperplasia cannot be discriminated from follicular hyperplasia using antibodies to PCNA or to nerve growth factor receptor.

Monoclonal antibodies against the 43,000 da glycoprotein from Paracoccidioides brasiliensis modulate laminin-mediated fungal adhesion to epithelial cells and pathogenesis

The surface glycoprotein gp43, a highly immunogenic component of Paracoccidioides brasiliensis, is used in the serodiagnosis of paracoccidioidomycosis (PCM) and has recently been shown to specifically bind the extracellular matrix protein laminin, Binding to laminin induces the increased adhesion of the fungus to epithelial cells; a hamster testicle infection model has shown that the gp43-dependent binding of fungal cells to laminin enhances their pathogenicity in vivo. We report on the production and characterization of 12 monoclonal antibodies against the gp43 that recognize peptide sequences in the molecule detecting at least three different epitopes as well as different isoforms of this antigen. MAbs interfered in the fungal pathogenicity in vivo either by inhibiting or enhancing granuloma formation and tissue destruction, Results suggest that P. brasiliensis propagules may start infection in man by strongly adhering to human lung cells, Thus, laminin-mediated fungal adhesion to human lung carcinoma (A549) cells was much more intense than to Madin-Darby canine kidney cells (MDCK), indicating differences in binding affinity, Subsequent growth of fungi bound to the lung cells could induce the granulomatous inflammatory reaction characteristic of PCM. Both steps are greatly stimulated by laminin binding in infective cells expressing gp43.

Thyroid cell proliferation in Graves' disease - Use of MIB-1 monoclonal antibody

OBJECTIVE: To measure thyroid cell proliferation in patients with Graves' disease (GD) before and during treatment with antithyroid drugs.STUDY DESIGN: Patients were assessed by fine needle aspiration biopsy before (n=20) and after 4 (n=19) and 12 months of treatment (n=15) with propylthiouracil or methimazole. Cell proliferation index (CPI) was estimated by immunocytochemistry using MIB-1. CPI was studied in relation to the cytologic parameters of the smears; clinical parameters, such as Wayne's Clinical Index (WCI) and time without treatment; laboratory parameters, such as (131)Iuptake and dosage of serum free thyroxin and thyroid-stimulating hormone; and thyroid ultrasound.RESULTS: CPI varied from 0.00% to 25.00% before treatment, 0.00% to 23.00% at 4 months and 0.00% to 14.84% at 12 months. CPI median values were 6.50%, 4.30% and 3.30%, respectively (before and after 4 months and 12 months of treatment). CPI had a positive correlation with WCI and FT4 at 12 months of treatment.CONCLUSION: Thyroid CPI in GD varies from case to case. However, due to its decreasing pattern during follow-up and its positive correlation with thyrotoxicosis severity, CPI may indicate the functional status of the gland and contribute to a better understanding of GD.

Estudo comparativo entre os métodos LSAB®+ e Herceptest® para a detecção de HER-2/neu em carcinoma de mama

INTRODUÇÃO: A hiperexpressão de human epidermal growth factor receptor (HER-2/neu) e a amplificação do seu gene são indicadores de formas mais agressivas do câncer de mama. A Food and Drug Administration (FDA) aprovou o teste denominado HercepTest® com a finalidade de selecionar pacientes com indicação para o uso de um anticorpo humanizado anti-HER-2/neu (trastuzumab), com efeito terapêutico comprovado. OBJETIVOS: O presente trabalho tem como objetivo comparar os resultados obtidos pelos métodos imuno-histoquímicos LSAB®+ com a utilização de anticorpo A0485 e HercepTest®. Material e métodos: Foram utilizados 50 casos de carcinoma de mama nos quais a pesquisa da hiperexpressão de HER-2 pelo método LSAB®+ já havia sido realizada. Foi repetida a pesquisa da hiperexpessão de HER-2/neu nos mesmos casos, utilizando-se o método do HercepTest®. RESULTADOS: 34 casos foram considerados negativos pelos dois métodos, com escore 0 pelo método HercepTest®. Destes, 12 obtiveram escore 1+ e 22 obtiveram escore 0 pelo método LSAB®+. em oito casos, o escore foi 2+ pelos dois métodos. Escore 3+ foi encontrado também em oito casos pelos dois métodos. DISCUSSÃO: O método mais prático utilizado em laboratório de rotina diagnóstica para investigar a hiperexpressão de HER-2/neu é o estudo imuno-histoquímico. em função de muitas variáveis, como tempo de fixação, tipo de fixador, duração da fixação, método de recuperação antigênica e tipo de anticorpo utilizado, pode haver divergência de resultados. CONCLUSÃO: O presente estudo concluiu que o método HercepTest®demonstrou resultados equivalentes aos resultados obtidos pelo método LSAB®+ em carcinoma de mama.

Obtenção e caracterização de anticorpo monoclonal murino anti-fator VIII da coagulação sangüínea

Entre os avanços da engenharia celular e biotecnologia nas últimas décadas, destaca-se a produção de anticorpos monoclonais murinos (AcMm) utilizados no aprimoramento diagnóstico nas rotinas laboratoriais. A produção de fator VIII de alta pureza sempre foi o desejo e a preocupação das indústrias de hemoderivados para tratamento de pacientes portadores de hemofilia A, porém este produto inexiste no Brasil, sendo necessária sua obtenção no mercado internacional a custos elevados. O trabalho tem por objetivo a produção de AcMm anti-fator VIII humano (FVIII H) através da expansão dos clones e caracterização imunoquímica do anticorpo. Camundongos Balb/c foram imunizados com FVIII H purificado como também proveniente de crioprecipitado e as células esplênicas dos animais foram fusionadas com células mielomatosas murinas segundo o método descrito por Kohler e Milstein para produção de híbridos em cultura. Foram testados 1.983 híbridos dos quais 105 foram submetidos à clonagem. Destes, 39 obtiveram monoclonalidade e 7 destes clones foram caracterizados através de técnicas de immunoblotting. Foram submetidas à purificação por cromatografia três imunoglobulinas de diferentes classes pertencentes aos clones LAMB1-10A1A4, LAMB1-17A1A1 e LAMB1-24A2A1. A imunoglobulina purificada pertencente ao clone LAMB1-10A1A4 foi adsorvida em coluna de imunoafinidade para purificação de concentrado de FVIII proveniente de crioprecipitado plasmático.

Detection and localization of small nuclear ribonucleoproteins (snRNPs) in trypanosomatids using anti-m(3)G antibodies

This work reports for the first time the identification and immunolocalization, by confocal and conventional indirect immunofluorescence, of m(3)G epitopes present in ribonucleoproteins of the following trypanosomatids: Trypanosoma cruzi epimastigotes of three different strains, Blastocrithidia ssp., and Leishmania major promastigotes. The identity of these epitopes and hence the specificity of the anti-m(3)G monoclonal antibody were ascertained through competition reaction with 7-methylguanosine that blocks the Ig binding sites, abolishing the fluorescence in all the parasites tested and showing a specific perinuclear localization of the snRNPs, which suggests their nuclear reimport in the parasites. Using an immunoprecipitation technique, it was also possible to confirm the presence of the trimethylguanosine epitopes in trypanosomatids.

IgG subclass profile of serum antibodies to Leishmania chagasi in naturally infected and vaccinated dogs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Monoclonal Antibody Production and Immunolocalization of a Salinity Stress-Related Protein in Rice (Oryza sativa)

Among various physiological responses to salt stress, the synthesis of a lectin-related protein of 14.5 kDa was observed in rice plants (Oryza sativa L.) under the treatment of 170 mmol/L NaCl. In order to better understand the role of the SALT protein in the physiological processes involving salinity, it was immunolocalized in mesophilic cells of leaf sheath and blade of a rice variety IAC-4440 following monoclonal antibodies produced by hybridome culture technique. This variety turned out to be an excellent model for that purpose, since it accumulates SALT protein even in absence of salt treatment and it has been classified as moderately sensitive to salinity and a superior grain producer. This feature was relevant for this work since it allowed the use of plants without the deleterious effects caused by salinity. Immunocytochemistry assays revealed that the SALT protein is located in the stroma of chloroplasts under non-stressing condition. Since the chloroplast is the main target affected by salinity and considering that the SALT protein does not present any apparent signal peptide for organelle localization, its lectin-like activity seems to play an important role in the establishment of stable complexes, either to other proteins or to oligosaccharides that are translocated to the chloroplast. © 2011 China National Rice Research Institute.

Caracterização e validação de anticorpo monoclonal murino anti-Linfócitos B humanos para uso em citometria de fluxo e imunoquímica

Pós-graduação em Pesquisa e Desenvolvimento (Biotecnologia Médica) - FMB

Validação de anticorpo monoclonal anti-CD45 obtido in house para utilização em citometria de fluxo e imuno-histoquímica

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)