Imunologia – 2ª parte Aplicações práticas (alguns exemplos) Anticorpos Monoclonais Citometria de fluxo Com ou sem anticorpos monoclonais Métodos em Alergologia Basotest Determinação de IgE Alergograma CAST

Anticorpos monoclonais Anticorpos produzidos apenas por um clone de células Especificidade Reprodutibilidade

Aplicações dos Ac monoclonais Identificação e doseamentos Diagnóstico Humoral Fenotipagem celular / contagem Quantificação de proteínas em amostras Terapêutica Anti-neoplásica Anti-inflamatória Outras

Populações linfocitárias

Populações linfocitárias

Populações linfocitárias

TESTE DE ACTIVAÇÃO DE BASÓFILOS marcador directo de activação celular CD 63 / CD 203c marcador indirecto de libertação de histamina Apresenta boa sensibilidade e especificidade B

TESTES DE ACTIVAÇÃO DE BASÓFILOS CITOMETRIA DE FLUXO Apreciação de reacções IgE mediadas Determinação da percentagem de basófilos activados Estimulação “in vitro” com alergénios específicos Boa correlação com outros testes “in vitro” e “in vivo” SPAIC-OUTUBRO /2003

Testes Laboratoriais IgE _______________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

IgE TOTAL _______________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

IgE TOTAL _______________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

UNICAP

IgE Total IgE Específica Rast ImunoCAP Ac anti-IgE marcado IgE sérica Fase sólida com Ac anti-IgE _______________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________ ImunoCAP

Multi-Rast Phadiatop ImunoCAP _______________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________ ImunoCAP

Rabo de Gato Phleum Pratense Extracção ____________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

Rabo de Gato Phleum Pratense Extracção ____________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________ Padrão proteico de doentes alérgicos por immunobloting

Allergograma (Phleum pratense) Rabo de Gato Phleum Pratense Phl p1 Phl p5

Alergénios recombinantes -técnica poly-(A) Allergy-eliciting biological material Standard Allergen ImmunoCAP Extraction, Immobilization on solid phase mRNA extraction poly-(A) cDNA synthesis, cloning and selection of cDNA clones encoding individual allergenic proteins Recombinant Allergen Production of recombinant allergens, Immobilization on solid phase Standardization and research reagents Pharmacia-Diagnostics

“MICROARRAY” DE ALERGÉNIOS: TÉCNICA Clin Exp. Allergy 2003; 7-13 GAILL-2003

DIAGNÓSTICO DA ALERGIA (Presente) Valenta,R. et all, Clinical Experimental Allergy, 1999, vol 29, 896-904 GAILL-2003

ALERGÉNIOS RECOMBINANTES-DIAGNÓSTICO Valenta,R. et all, Clinical Experimental Allergy, 1999, vol 29, 896-904 GAILL-2003

Cellular Antigen Stimulation Test Whole-blood EDTA Dextran sedimentation and 90 minutes incubation Transfer leukocytes, centrifuge, remove/discard upper phase + stimulation buffer (IL-3) sLT sLT A A sLT A + Antigen A A A A A A Supernatants can be harvested, after centrifugation store at - 20 °C until ELISA test Leukocyte suspension, + antigen (40 min incubation)

Cellular Antigen Stimulation Test 2. ELISA-Procedure Enzym sLT sLT sLT sLT Enzym sLT sLT sLT 2 h incubation, wash 3 times Supernatant + monoclonal antibody + substrate Enzym Color Reading at 405 nm sLT sLT sLT 30 min incubation, wash 3 times + stop-solution

Cellular Antigen Stimulation Test (CAST) Disponível para Antibióticos: Penicilina, Ampicilina /Amoxicilina Cefalosporina, Cefuroxime Trimethoprim, Sulfametoxazol Tetraciclina Ciprofloxacina Analgésicos: Aspirina, Diclofenac, Ibuprofeno, Indometacina Paracetamol, Metamizol Anestésicos Miorelaxantes

Imunologia – 2ª parte Aplicações práticas (alguns exemplos) Anticorpos Monoclonais Citometria de fluxo Com ou sem anticorpos monoclonais Métodos em Alergologia Basotest Determinação de IgE Alergograma CAST