<p>This Master Thesis was conducted at Octapharma AB in Stockholm.</p><p>Anticomplementary activity (ACA) is a measure of the product’s abilities to activate the complement system. IgG aggregates are mainly responsible for this activation. Two different performances of a method for determination of ACA in Octagam^® are available. The two performances are based on the reference method for test of ACA in immunoglobulins in the European Pharmacopoeia Commission Guideline 6.0 (chapter 2.6.17). The method is carried out either in test tubes or on microtiter plates. The test tube method can be performed either in a manual manner or modified, being more automated. The latter performance has been applied in this study. The plate method is more automated than both of the tube methods. The plate method and the manual tube method have earlier seemed to result in different outcomes, which was the basis for this thesis.</p><p>The plate method and the modified test tube method have been compared and robustness parameters have been studied in order to see which factors influence on the end result. The adequacy of using Human Biological Reference Preparation (human BRP) as a control for the ACA method in general has also been investigated. Samples of the product are outside the scope of this thesis and have not been investigated.</p><p>According to this study, the plate method and the modified tube method are not comparable with regard to complement titration results and to ACA of the BRP control. A higher precision is gained with the plate method. This in combination with the higher degree of automation makes the plate method advantageous in several aspects. When it comes to the robustness of the ACA method in general, the sheep red blood cells (SRBC) used are critical. Haemolysin dilution and complement activity seem to be critical as well.</p><p>Human BRP is, according to this study more adequate as a reference for the plate method than for the tube method. An In house control is believed to be more representative to the ACA method in general as it is of the same nature as the samples analysed, in contrast to the human BRP.</p>

Made available in DSpace on 2017-07-10T17:48:08Z (GMT). No. of bitstreams: 1 Thiago_Andrade.pdf: 1549931 bytes, checksum: 1f4ef56f0d7a56140c61a2c47da50b4d (MD5) Previous issue date: 2015-04-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Two experiments were conducted in order to achieve the effect of enzymes in diet based on corn and soybean meal. The first experiment aimed to evaluate the performance, carcass yield and ileal digestibility in broilers fed enzymes 21-42 days old. They were used in 1008 broiler chickens, Cobb-500, males, distributed in a completely randomized design involving six treatments and eight repetitions defined as follows: 1) Food positive control (CP) based on corn and soybean meal; 2) negative control diet (CN) based on corn and soybean meal with a reduction of 120 kcal / kg metabolizable energy (ME); 3) EC1 (CN + adding 100 ppm of Econase); 4) CE2 (CN + addition of 200 ppm of Avizyme); 5) BE1 (CN + addition of 100 ppm and 200 ppm xylanase Amylase); 6) BE2 (CN + with addition of 100 ppm 300 ppm xylanase and amylase). The data on performance, income cuts, coefficient and nutrient digestibility were analyzed using the boxplot test (SAS, 2002), and after the removal of outliers was performed ANOVA and subsequent average test (Tukey) to the level of 5% probability. As a result, there was no significant effect on feed intake. However, the results observed with CE2 and BE2 enzymes in weight gain variables, final weight, feed conversion, digestibility of dry matter, crude protein, crude energy and nutrient digestibility of dry matter, crude protein and energy Gross obtained results similar to CP. Concluding that the DM digestibility coefficients, PB and ED, digestibility of DM, CP, EB, GP data, PF, CA and carcass yield, the enzyme complex 2 (CE2) and blend enzyme 2 (BE2 ), when added to the diet to reduce energy provided similar results to the positive control. The second experiment was to evaluate the performance, carcass yield, cuts and blood test in broilers fed enzyme complexes 01-42 days old. They were used 960 broiler, Cobb-500, males, distributed in a completely randomized design involving six treatments and eight repetitions defined as follows: 1) Food positive control (CP) based on corn and soybean meal; 2) feed negative control (CN) based on corn and soybean meal with a reduction of 5% in the amino acid values, protein and metabolizable energy (ME); 3) BE1 (CN + addition of 125 ppm Poultrygrow); 4) BE2 (CN + addition of 100 ppm of axtra); 5) BE3 (CN + addition 500 ppm Ronozyme); 6) BE4 (CN + addition of 125 ppm and 500 ppm Poultrygrow Hemicell). We evaluated performance (feed intake, weight gain and feed conversion), yield (carcass and cuts), and blood tests (cholesterol, triglycerides, total protein, uric acid, creatinine, alanine aminotransferase, aspartate aminotransferase, albumin, glucose, urea). Performance data, carcass yield, cuts, organs, abdominal fat and blood parameters were submitted to boxplot test for removal of outliers, and subsequently was conducted analysis of variance and mean comparison test (Tukey) at 5% probability. The feed intake showed no significant difference between treatments. 7 of weight gain; 14; 21:35 days old CN and observed by the enzymes in improved weight gain, however, CP showed the best result. For the period 1-42 days just the CP met the aexigências of birds. Feed conversion in the pre-initial phase (1-7 days old) was positive (p <0.05) in CP. In the periods 1 to 14; 1 to 21 and 1 to 35 days of age, the enzyme supplementation gave similar results to the CN and CP value greater (p <0.05). Between 1-42 days of age, the CP had the best feed conversion, and the enzymes were similar to CN. The chickens fed diet enzyme provided superior result to the CP and similar to CN. The yield breast and wing showed no significant differences between treatments. The yield leg (thigh and drumstick) showed similar results between the enzyme and the CP. The yield livers and gizzards showed no significant difference and the relative pancreas weight showed enzyme gave rise compared with the diets of CN and CP. The abdominal fat yield observed in the CN and enzymatic treatments provided by increasing the amount of abdominal fat compared to CP. The result of biochemical blood cholesterol profile indicated that the enzyme supplementation can influence the increase in cholesterol levels, being observed greater value in CE3. The triglyceride level observed in enzymatic treatments was similar to CP, indicating the action of enzymes in the diet. Albumin variable demonstrated that the addition of the enzyme complex was not sufficient. ALT levels demonstrated that the enzymatic complex and yielded low activity CN, the CP and the highest level. The levels of glucose, total protein and aspartate amino transferase (AST), creatinine and uric acid were similar for all treatments and presented according to the values recommended for lineage. Concluding that nutritional reductions in poultry diets affect performance, carcass characteristics and biochemical profile and that supplementation of enzymatic complexes was not efficient in the release of nutrients when using matrix with severe reductions / Dois experimentos foram conduzidos com o intuito de realizar o efeito de enzimas em dieta a base de milho e farelo de soja. O primeiro experimento teve como objetivo avaliar o desempenho, rendimento de carcaça e digestibilidade ileal em frangos de corte alimentados com enzimas de 21 a 42 dias de idade. Foram utilizados 1008 frangos de corte, Cobb-500, machos, distribuídos em um delineamento inteiramente casualizado envolvendo seis tratamentos e oito repetições, definidos assim: 1) ração controle positivo (CP) a base de milho e farelo de soja; 2) ração controle negativo (CN) a base de milho e farelo de soja com redução de 120 kcal/kg de energia metabolizável (EM); 3) CE1 (CN + adição de 100 ppm de Econase); 4) CE2 (CN + adição de 200 ppm de Avizyme); 5) BE1 (CN + adição de 100 ppm de xilanase e 200 ppm de Amilase); 6) BE2 (CN + com adição de 100 ppm de xilanase e 300 ppm de amilase). Os dados obtidos no desempenho, rendimento, cortes, coeficiente e digestibilidade de nutrientes foram analisados através do teste de boxplot (SAS, 2002), e, após a retirada dos outliers, foi realizada análise de variância e posterior teste de média (Tukey) ao nível de 5% de probabilidade. Como resultado, não foi observado efeito significativo no consumo de ração. No entanto, o resultado observado com as enzimas CE2 e BE2 nas variáveis de ganho de peso, peso final, conversão alimentar, coeficiente de digestibilidade da matéria seca, proteína bruta, energia bruta e da digestibilidade de nutrientes da matéria seca, proteína bruta e energia bruta apresentou semelhanças ao CP. Concluindo que os coeficientes de digestibilidade da MS, PB e ED, digestibilidade dos nutrientes da MS, PB, EB, os dados de GP, PF, CA e ao rendimento de carcaça, o complexo enzimático 2 (CE2) e blend enzimático 2 (BE2), quando adicionados à dieta com redução energética proporcionaram resultado semelhante ao controle positivo. O segundo experimento teve como objetivo avaliar o desempenho, rendimento de carcaça, cortes e análise de sangue em frangos de corte alimentados com complexos enzimáticos de 01 a 42 dias de idade. Foram utilizados 960 frangos de corte, Cobb-500, machos, distribuídos em um delineamento inteiramente casualizado envolvendo seis tratamentos e oito repetições definidos assim: 1) ração controle positivo (CP) a base de milho e farelo de soja; 2) ração controle negativo (CN) a base de milho e farelo de soja com redução de 5% nos valores de aminoácido, proteína e energia metabolizável (EM); 3) BE1 (CN + adição de 125 ppm de Poultrygrow); 4) BE2 (CN + adição de com 100 ppm de Axtra); 5) BE3 (CN + adição de 500 ppm de Ronozyme); 6) BE4 (CN + adição de 125 ppm de Poultrygrow e 500 ppm de Hemicell). Os parâmetros avaliados foram de desempenho (consumo de ração, ganho de peso e conversão alimentar), rendimento (carcaça e cortes), e análise de sangue (colesterol, triglicerídeos, proteína total, ácido úrico, creatinina, alanina aminotransferase, aspartato aminotransferase, albumina, glicose, ureia). Os dados de desempenho, rendimento de carcaça, cortes, órgãos, gordura abdominal e os parâmetros sanguíneos foram submetidos ao teste de boxplot para retirada dos outliers e, posteriormente, foi realizada análise de variância e teste de média (Tukey) ao nível de 5% de probabilidade. O consumo de ração não apresentou diferença significativa entre os tratamentos. O ganho de peso de 7; 14; 21 e 35 dias de idade, observado no CN, e pelas enzimas melhoraram o ganho de peso, contudo, CP apresentou o melhor resultado. Para o período de 1 a 42 dias apenas o CP atendeu as a exigências das aves. A conversão alimentar na fase pré-inicial (1-7 dias de idade) apresentou resultado positivo (p<0,05) apenas no CP. Nos períodos de 1 a 14; 1 a 21 e 1 a 35 dias de idade, a suplementação enzimática proporcionou resultado semelhante ao CN e o CP o maior valor (p<0,05). No período de 1 a 42 dias de idade, o CP apresentou a melhor conversão alimentar, e as enzimas foram semelhantes ao CN. Os frangos alimentados com dieta enzimática proporcionaram resultado superior ao CP e semelhante ao CN. O rendimento de peito e asa não apresentaram diferenças significativas entre os tratamentos. O rendimento de perna (coxa e sobrecoxa) apresentou resultado semelhante entre as enzimas e o CP. O rendimento de fígado e moela não apresentou diferença significativa e o peso relativo do pâncreas de frangos foi influenciado pelos tratamentos enzimáticos quando comparados as aves de CP e CN. O rendimento de gordura abdominal observada no CN pelos tratamentos enzimáticos proporcionou aumento na quantidade de gordura abdominal quando comparado com CP. O resultado do perfil bioquímico sanguíneo do colesterol indicou que a suplementação enzimática pode influenciar no aumento dos teores do colesterol, sendo observado maior valor no CE3. O nível de triglicerídeos observados nos tratamentos enzimáticos foi semelhante ao CP, indicando a ação das enzimas na dieta. A variável de albumina demonstrou que a adição dos complexos enzimáticos não foi suficiente. Os níveis de ALT demonstrou que os complexos enzimáticos e o CN proporcionaram baixa atividade, e o CP o maior nível. Os valores de glicose, proteína total e aspartato amino-transferase (AST), creatinina e ácido úrico foram semelhantes em todos os tratamentos e apresentaram valores conforme o recomendado para linhagem. Como resultado, tem-se que as reduções nutricionais nas dietas de aves afetam o desempenho, características de carcaça e perfil bioquímico e, também, que a suplementação dos complexos enzimáticos não foi eficiente na liberação dos nutrientes quando utilizada matriz com severas reduções

This Master Thesis was conducted at Octapharma AB in Stockholm. Anticomplementary activity (ACA) is a measure of the product’s abilities to activate the complement system. IgG aggregates are mainly responsible for this activation. Two different performances of a method for determination of ACA in Octagam® are available. The two performances are based on the reference method for test of ACA in immunoglobulins in the European Pharmacopoeia Commission Guideline 6.0 (chapter 2.6.17). The method is carried out either in test tubes or on microtiter plates. The test tube method can be performed either in a manual manner or modified, being more automated. The latter performance has been applied in this study. The plate method is more automated than both of the tube methods. The plate method and the manual tube method have earlier seemed to result in different outcomes, which was the basis for this thesis. The plate method and the modified test tube method have been compared and robustness parameters have been studied in order to see which factors influence on the end result. The adequacy of using Human Biological Reference Preparation (human BRP) as a control for the ACA method in general has also been investigated. Samples of the product are outside the scope of this thesis and have not been investigated. According to this study, the plate method and the modified tube method are not comparable with regard to complement titration results and to ACA of the BRP control. A higher precision is gained with the plate method. This in combination with the higher degree of automation makes the plate method advantageous in several aspects. When it comes to the robustness of the ACA method in general, the sheep red blood cells (SRBC) used are critical. Haemolysin dilution and complement activity seem to be critical as well. Human BRP is, according to this study more adequate as a reference for the plate method than for the tube method. An In house control is believed to be more representative to the ACA method in general as it is of the same nature as the samples analysed, in contrast to the human BRP.