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Detection of recombinant human Erythopoietin and analogues through immunorecognition and n-glycolyl-neuraminic acid identificationMallorquí Bagué, Joaquim 28 April 2011 (has links)
Erythropoietin (EPO) is a glycoprotein hormone, the molecule comprises a single polypeptide chain of 165 aminoacids with two disulfide bonds, 1 O-linked (Ser-126), and 3 N-linked (Asn-24, 38, 83) glycans representing about 40 % of the total mass (30 kDa). It is secreted primarily by adult kidneys in response to tissue hypoxia and it is involved in the maturation and ultimately regulation of the level of red blood cells. The recombinant analogue (rhEPO), available since 1989 has found widespread use in the treatment of different diseases. Besides, rhEPO is illicitly used by athletes to boost the delivery of oxygen to the tissue and enhance performance in endurance sports. Current tests to differentiate between endogenous EPO and its recombinant analogues are based on differences in their isoelectric focussing (IEF) profiles and on differences in their molecular weight (SDS-PAGE). In this study, different methods to facilitate the detection of recombinant EPOs and analogues in antidoping control have been developed: A plasmatic EPO immunopurification method; a new screening method based on immunoaffinity techniques to detect the abuse of recombinant erythropietins in urine; and a liquid chromatography-mass spectrometry method that allows to detect the unambiguous differing structure between exogenous EPOs and endogenous, the N-glycolyl-neuraminic acid. / La eritropoetina (EPO) és una hormona glicoproteica formada per una cadena peptídica de 165 aminoàcids que conté dos ponts disulfur, un O-glicà (Ser-126) i tres N-glicans (Asn-24, 38, 83) que representen al voltant d’un 40% de la seva massa molar (~ 30kDa). Es produeix principalment en el ronyó, en resposta a la reducció d’oxigen en el teixits, i estimula l’eritropoesi a la medul·la òssia. La EPO recombinant (rhEPO) s’administra com a fàrmac pel tractament de diferents malalties. També s’ha observat la seva utilització en esportistes amb l’objectiu d’augmentar el nivells d’oxigen als teixits i així incrementar el seu rendiment. Els mètodes que s’utilitzen per diferenciar la EPO orinaria endògena de l’exògena estan basats en diferencies dels seus perfils isoelectroforètics (IEF) o en els seus pesos moleculars (SDS-PAGE). El problema d’aquests mètodes és que són llargs, costosos i només poden utilitzar la orina com a matriu biològica. En aquest estudi, s’ha dut a terme el desenvolupament dels següents mètodes que faciliten la detecció d’EPOs recombinants i anàlegs en el control antidopatge: Un mètode d’immunopurificació d’EPO en plasma; un mètode d’screening ràpid basat en tècniques d’immunoafinitat per detectar l’abús d’ eritropoietines recombinants en orina; i un mètode de cromatografia liquida acoblada a espectrometria de massa que permet detectar una clara diferencia estructural entre la majoria de les EPOs exògenes i la endògena, el N-glicolil-neuraminic àcid.
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Anticorpos anti-Stx como ferramentas na detecção de Escherichia coli produtora da toxina de Shiga (STEC). / Antibodies anti-Stx as tools on detection of Shiga toxin-producing Escherichia coli (STEC).Rocha, Letícia Barboza 15 April 2008 (has links)
Infecções causadas por STEC constituem problema de saúde pública, pois estão associadas à síndrome hemolítica urêmica e colite hemorrágica. A detecção de isolados STEC depende de um ensaio diagnóstico sensível, específico e de baixo custo. Neste sentido, o presente estudo teve como objetivo o desenvolvimento de um ELISA de captura utilizando anticorpos policlonais e monoclonais. Primeiramente definiu-se um meio de cultivo para maior expressão de Stx e produziu-se anticorpos monoclonais anti-Stx1 e anti-Stx2. Os resultados mostraram que o cultivo de isolados por 4h em caldo EC com ciprofloxacina induziu maior produção de Stx no sobrenadante bacteriano, e o anticorpo monoclonal anti-Stx1 produzido reconheceu a subunidade A de ambas as toxinas, além de apresentar maior constante de afinidade que o anti-Stx2. Desta forma, o ELISA de captura foi padronizado com 250 mg/ml da fração IgG do soro policlonal anti-Stx1 e anti-Stx2 na sensibilização e 2,5 mg/ml de anticorpo monoclonal anti-Stx1 na captura da toxina, apresentando 80,7% de sensibilidade e 100% de especificidade. / STEC infection is a public health problem, causing hemolytic-uremic syndrome and haemorrhagic colitis. The detection of STEC isolates depends on a sensitive, specific and low cost diagnostic method. Thus, the aim of this study was to develop a capture ELISA using polyclonal and monoclonal antibodies. For this purpose, first of all a growth media that induce better expression and production of Stx was defined and monoclonals anti-Stx1 e anti-Stx2 antibodies were produced. The results show that the growth of the isolates for 4hs in EC broth with ciprofloxacin increased the production of Stx in the bacterial supernantant, and the monoclonal anti-Stx1antibody produced was able to recognize the A subunit of both toxins, besides showing higher affinity constant than anti-Stx2. Thus, the capture ELISA was standardized using 250 mg/ml of the IgG enriched fraction of rabbit anti-Stx1 sera and anti-Stx2 for coating and 2,5 mg/ml of monoclonal anti-Stx1 antibody for the capture of the toxin, showing 80,7% of sensibility and e 100% de specificity.
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Anticorpos anti-Stx como ferramentas na detecção de Escherichia coli produtora da toxina de Shiga (STEC). / Antibodies anti-Stx as tools on detection of Shiga toxin-producing Escherichia coli (STEC).Letícia Barboza Rocha 15 April 2008 (has links)
Infecções causadas por STEC constituem problema de saúde pública, pois estão associadas à síndrome hemolítica urêmica e colite hemorrágica. A detecção de isolados STEC depende de um ensaio diagnóstico sensível, específico e de baixo custo. Neste sentido, o presente estudo teve como objetivo o desenvolvimento de um ELISA de captura utilizando anticorpos policlonais e monoclonais. Primeiramente definiu-se um meio de cultivo para maior expressão de Stx e produziu-se anticorpos monoclonais anti-Stx1 e anti-Stx2. Os resultados mostraram que o cultivo de isolados por 4h em caldo EC com ciprofloxacina induziu maior produção de Stx no sobrenadante bacteriano, e o anticorpo monoclonal anti-Stx1 produzido reconheceu a subunidade A de ambas as toxinas, além de apresentar maior constante de afinidade que o anti-Stx2. Desta forma, o ELISA de captura foi padronizado com 250 mg/ml da fração IgG do soro policlonal anti-Stx1 e anti-Stx2 na sensibilização e 2,5 mg/ml de anticorpo monoclonal anti-Stx1 na captura da toxina, apresentando 80,7% de sensibilidade e 100% de especificidade. / STEC infection is a public health problem, causing hemolytic-uremic syndrome and haemorrhagic colitis. The detection of STEC isolates depends on a sensitive, specific and low cost diagnostic method. Thus, the aim of this study was to develop a capture ELISA using polyclonal and monoclonal antibodies. For this purpose, first of all a growth media that induce better expression and production of Stx was defined and monoclonals anti-Stx1 e anti-Stx2 antibodies were produced. The results show that the growth of the isolates for 4hs in EC broth with ciprofloxacin increased the production of Stx in the bacterial supernantant, and the monoclonal anti-Stx1antibody produced was able to recognize the A subunit of both toxins, besides showing higher affinity constant than anti-Stx2. Thus, the capture ELISA was standardized using 250 mg/ml of the IgG enriched fraction of rabbit anti-Stx1 sera and anti-Stx2 for coating and 2,5 mg/ml of monoclonal anti-Stx1 antibody for the capture of the toxin, showing 80,7% of sensibility and e 100% de specificity.
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