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Febre
Estudos h + 2000 anos funo teraputica
1884: GRAM
Febre
1912: Hort e Penfold esterilizao trmica ou
filtrao no eliminava a febre das injees
Pirognios
Pirognio exgeno (PEx): endotoxinas e outros de origem microbiana ou no.
Pirognio endgeno (Pen): produzido em resposta ao Pex Mediador primrio da febre (~15.000 daltons) Produo de PGs
Endotoxina X LPS
LPS
Ao biolgica
Estrutura geral do LPS de Salmonella : Glc = glicose; Gal = galactose; Etn = etanolamina; GlcNac = N-acetil- glicosamina; Hep = heptose; P = fosfsto; R1 and R2 = fosfoetanolamina ou aminoarabinose.
Antigenicidade
Disperso
Endotoxinas
Ao biolgica (Lipdio A): Toxicidade letal
Leucopenia leucocitose Necrose da medula ssea PA Agregao plaquetria Induo de resistncia no especfica infeco Atividade de macrfagos Induo produo de interferon Induo produo de fator necrtico Hipotermia em camundongos Gelificao do amebcito de Limulus Etc. etc.
Endotoxinas
Mecanismo de induo: Fagocitose lipdio A produo de PEn Travessia da BH-E
Ao hipotalmica Lipdio A tb. age no crebro Ratos e camundongos X Humanos e coelhos
No hipertermia Lipdio A ou PEn no atravessam BH-E Hipotermia ao vascular perifrica
Outros Pirognios
Exotoxinas Enterotoxinas Outros mo. Substncias em geral
2) Destilao 3) Ultrafiltrao 4) Osmose Reversa 5) Carvo ativado 6) Atrao (eletrosttica / por membrana hidrofbica)
Pirognios
Coelhos: qualitativa e quantitativamente semelhante ao ser humano. ~1,5 Kg (injeo de 10mL/Kg para gdes volumes tempo excessivo) Fator de segurana Raas pequenas Triagem Reutilizao dos animais Descanso Ateno para doses sub-pirognicas
Teste de Pirognios
Requisitos animal:
Mesmo sexo Adultos Sadios Mesma raa (preferencialmente) Peso mnimo: 1,5 Kg
Teste de Pirognios
Condicionamento:
Ao menos 1 vez At 7 dias antes teste
Se temperatura 0,5C X
Teste:
Animais com T 39,8C Variao interindividual 1,0C
Teste de Pirognios
Registro T:
Preciso 0,1C Insero retal (~6 cm)
Procedimento prvio:
Suspenso alimentao 2 h antes Ambiente: No stress (Dont worry. Pode piorar) Registro T mx 40 min. antes (2 leituras a cada 30
min.) mdia a T controle
Teste de Pirognios
Injeo:
Produto a 37,0 2,0C Em 3 coelhos Veia marginal 0,5mL Volume injetado 10 mL / Kg (ou de
acordo com monografia)
Teste de Pirognios
Interpretao:
Decrscimos desconsiderados
Teste de Pirognios
LAL
Limulus polyphemus
LAL
Tachypleus tridentatus
LAL
Limulus polyphemus
LAL
Limulus polyphemus
Salvem os ferradurinhas!!!!!
The Lorch Foundation Detection of Pyrogens HORSESHOE CRABS FUTURE SECURED BY NEW INVENTION NEW DETECTOR FOR PYROGENS IN ULTRA PURE WATER AND WATER FOR INJECTION SAVES USING BLOOD FROM ENDANGERED HORSESHOE CRABS.
Optical biosensor utilising Surface Plasmon Resonance technology. Detection of pyrogens (endotoxin) to below the FDA and Pharmacopoeia limits (0.25 EU/ml). European and United States Patents applied for (Priority date 29 July 1998). Potential to develop a continuous, real-time on line monitor. Eliminates the use of blood extracts from the rare and endangered horseshoe crab species, Limulus. Use in pharmaceutical and micro-electronic manufacturing as well as research and analytical laboratories. Obviates the need for batch testing. "The ideal solution to the problem of ensuring that water is apyrogenic would be an on-line monitor installed at the point of collection. The discovery of such an instrument has been the holy grail of pharmaceutical and ultra pure water industries for several decades ." Leonard Saunders, D.Sc., F.R.S.C., Emeritus Professor of Pharmaceutical Chemistry, University of London The Lorch Foundation in collaboration with Cranfield University's Institute of BioScience and Technology has developed a sensor for the detection of pyrogens in ultra pure water. This sensor will supersede the existing LAL test, an assay utilising blood extract from the Limulus Horseshoe Cra b. The new sensor can detect endotoxin down to the level of 0.01 EU/ml (Endotoxin units per millilitre), which is below the FDA requirements an d those of the United States and European Pharmacopoeias for Water for Injection. The existing LAL test has severe limitations in that it requires discreet batch sampling, must be carried out by trained technicians in a laboratory environment and cannot provide instant results. In order to obtain the material, the rare and endangered horseshoe crabs are fished from the ocean and either bled and returned or crushed and their future is limited. The Lorch Foundation is now seeking an exclusive commercial licensee for the final development and marketing of this patented technology. The new instrument will be developed to provide on-line continuous results utilising optical technology without the need for laboratory batch testing. The product will be used in pharmaceutical and micro-electronic manufacturing as well as in research and testing laboratories. A paper: DETECTION OF PYROGENS IN ULTRA-PURE WATER BY A SURFACE PLASMON RESONANCE BIOSENSOR (Judith A Taylor, Gary Barrett, Walter Lorch, David C Cullen) was published in the peer reviewed journal Analytical Letters, volume 35 Issue 2, 22 February 2002. This can be accessed from: - http://www.dekker.com/servlet/product/DOI/101081AL120002525#abstract
Vantagens EB X Pirog.
Rapidez - 60 min. X 180 min. Maior sensibilidade Menos variabilidade Menos falsos positivos Mais econmico
Teste LAL
Preparao para o teste Padro Endotoxina: 10000 UE (UI) / frasco Sol. Me: reconstituio com H2O grau LAL (5 mL)
Agitao por 30 min. Conservao por no mx.14 dias (refrigerao)
Validao por Testes de inibio e potencializao Controles negativos Sol. Amostra: diluies seriadas da sol. me aps agitao
Ajuste de pH (6,0 a 8,0) com tampes.
Teste LAL
Coagulao em tubo
1. Proenzima
Enzima Ativa
Enzima Ativa
2. Protena de coagulao
Cogulo
Teste LAL
Mtodos Fotomtricos
1. Proenzima
Enzima Ativa
Enzima Ativa
2. Substrato cromognico
COR
1. Proenzima
Enzima Ativa
Enzima Ativa
2. Protena de coagulao
TURBIDEZ
Testes cinticos
Testes Cinticos
Slope = -0.204 Y-Int = 3.058 R = -0.998