Effect of Lyophilization on Stability of PEG-Protein Conjugate: A Case Study with Peginterferon alfa-2b

Abstract The purpose of the present study was to develop stable lyophilized formulation of peginterferon alfa-2b which is acquiescent to the short lyophilization process. The present study evaluates the effect of buffering components and cryoprotectant(s) on depegylation of the peginterferon alfa-2b in combination with lyophilization process. Finally, a short lyophilization process was identified which can produce a stable pharmaceutical form of peginterferon alfa-2b without any depegylation during long-term storage. Formulations were analyzed mainly for depegylation by HP-size exclusion chromatography and in-vitro antiviral activity. Residual moisture content in the lyophilized product was also used as a key indicating parameter to check its role with respect to depegylation upon storage under various temperature conditions. It was observed that the peginterferon alfa-2b when formulated in presence of cryoprotectant like sucrose requires longer lyophilization process of about 5 days, irrespective of the buffering components used, to reduce the level of residual moisture content and thereby to produce the stable formulation without depegylation. A stable formulation in presence of high concentration of lactose as a cryoprotectant was developed which can withstand stresses exerted to protein-polymer conjugate during lyophilization phases without any significant depegylation. A short lyophilization process of about 48 hours can be utilized for peginterferon alfa-2b when formulated in presence of lactose as a cryoprotectant through which a stable lyophilized formulation can be produced as against longer process required when sucrose is used a cryoprotectant, which is essential from commercial point of view as lyophilization is a costly process.

Caracterización del polirribosilribitol fosfato por cromatografía de exclusión molecular de alta eficacia con detección ultravioleta / Characterization of polyribosylribitol phosphate by high efficiency size exclusion chromatography with ultraviolet detection

Haemophilus influenzae tipo b es un importante patógeno del hombre causante de varias de las enfermedades invasivas en niños menores de cinco años, contra el cual fueron autorizadas las vacunas glicoconjugadas a partir del polirribosilribitol fosfato. Quimi-Hib® es la primera y única vacuna contra este patógeno que utiliza el polisacárido obtenido por síntesis química. El Ingrediente Farmacéutico Activo es producido por el Centro de Ingeniería Genética y Biotecnología y se obtiene a partir de su conjugación al toxoide tetánico. En el presente reporte se hizo una caracterización del polirribosilribitol fosfato mediante la técnica de cromatografía de exclusión molecular de alta eficacia con detección ultravioleta a 215 nm. En el estudio se evaluaron tres lotes y se determinó el perfil de elución en una columna SuperdexTM 75 10/300 GL Increase con un porciento de pureza de 77,42 ± 8,97 y una masa molar promedio de 7.381 Da ± 210,93. La principal impureza presente en el polirribosilribitol fosfato es el dimetilsulfóxido, disolvente utilizado en la reacción de activación con el éster N-hidroxisuccinimidilo del ácido β-maleimidopropiónico. El polirribosilribitol fosfato se purificó por filtración con un Amicon Ultra-15 de 2.000 Da hasta una pureza de 99,1 por ciento y se conjugó al toxoide tetánico. El rendimiento de la reacción de conjugación con el polisacárido purificado fue de 30,0 por ciento 1,77 el cual no muestra diferencias significativas con el control que fue 33,7 por ciento ± 3,57 demostrándose que el dimetilsulfóxido no afecta el desempeño de la reacción de conjugación(AU)

Haemophilus influenzae type b is an important human pathogen causing some invasive diseases in children less than five years of age. Glycoconjugate vaccines based on polyribosylribitol phosphate have been licensed against this bacterium. Quimi-Hib® is the first and only vaccine against this pathogen using the chemically synthesized polysaccharide. The Active Pharmaceutical Ingredient is produced by the Center for Genetic Engineering and Biotechnology and is obtained from its conjugation to tetanus toxoid. In the present report a characterization of polyribosylribitol phosphate was performed by high performance molecular exclusion chromatography with ultraviolet detection at 215 nm. Three batches were evaluated in the study and the elution profile was determined on a SuperdexTM 75 10/300 GL Increase column with a purity percentage of 77.42 ± 8.97 and an average molecular weight of 7,381 Da ± 210.93. The main impurity present in polyribosylribitol phosphate was dimethylsulfoxide, the solvent used in the activation reaction with N-hydroxysuccinimidyl ester of β-maleimidopropionic acid. Polyribosylribitol phosphate was purified by filtration using a 2,000 Da cut-off Amicon Ultra-15 to a purity of 99.1 percent and conjugated to tetanus toxoid. The yield of the conjugation reaction with the purified polysaccharide was 30.0 percent ± 1.77 which shows no significant difference with the control which was 33.7 percent ± 3.57 demonstrating that dimethylsulfoxide does not affect the performance of the conjugation reaction(AU)

Retrovectors packaged in CHO cells to generate GLP-1-Fc stable expression CHO cell lines

Background: Chinese hamster ovary (CHO) cells are the most dependable mammalian cells for the production of recombinant proteins. Replication-incompetent retroviral vector (retrovector) is an efficient tool to generate stable cell lines. Multiple copies of integrated genes by retrovector transduction results in improved recombinant protein yield. HEK-293 and their genetic derivatives are principal cells for retrovector production. Retrovectors packaged in HEK-293 cells pose a risk of infectious agent transmission, such as viruses and mycoplasmas, from serum and packaging cells. Results: In this report, retrovectors were packaged in CHO cells cultured in chemically defined (CD) media. The retrovectors were then used to transduce CHO cells. This method can block potential transmission of infectious agents from serum and packaging cells. With this method, we generated glucagon-like protein-1 Fc fusion protein (GLP-1-Fc) stable expression CHO cell lines. Productivity of GLP-1-Fc can reach 3.15 g/L. The GLP-1-Fc protein produced by this method has comparable bioactivity to that of dulaglutide (Trulicity). These stable cell lines retain 95­100% of productivity after 40 days of continuous culture (~48­56 generations). Conclusions: Suspension CHO cells are clean, safe, and reliable cells for retrovector packaging. Retrovectors packaged from this system could be used to generate CHO stable cell lines for recombinant protein expression.

Quality of bevacizumab (Avastin®) repacked in single-use glass vials for intravitreal administration / Avaliação da qualidade do bevacizumabe (Avastin®) fracionado em frascos de vidro de dose única para administração intravítrea

ABSTRACT Purpose: Avastin® (bevacizumab) is an anti-vascular endothelial growth factor (VEGF) monoclonal antibody given as an off-label drug by intravitreal administration for treatment of ocular diseases. The drug's clinical application and its cost-benefit profile has generated demand for its division into single-use vials to meet the low volume and low-cost doses necessary for intraocular administration. However, the safety of compounding the drug in single-use vials is still under discussion. In this study, the stability and efficacy of Avastin® repacked in individual single-use glass vials and glass ampoules by external compounding pharmacies were evaluated. Methods: Polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatography (SEC), dynamic light scattering (DLS), and turbidimetry were selected to detect the formation of aggregates of various sizes. Changes in bevacizumab biological efficacy were investigated by using an enzyme-linked immunosorbent assay (ELISA). Results: Repacked and reference bevacizumab showed similar results when analyzed by PAGE. By SEC, a slight increase in high molecular weight aggregates and a reduction in bevacizumab monomers were observed in the products of the three compounding pharmacies relative to those in the reference bevacizumab. A comparison of repacked and reference SEC chromatograms showed that the mean monomer loss was ≤1% for all compounding pharmacies. Protein aggregates in the nanometer- and micrometer-size ranges were not detected by DLS and turbidimetry. In the efficacy assay, the biological function of repacked bevacizumab was preserved, with <3% loss of VEGF binding capacity relative to that of the reference. Conclusion: The results showed that bevacizumab remained stable after compounding in ampoules and single-use glass vials; no significant aggregation, fragmentation, or loss of biological activity was observed.

RESUMO Objetivos: Avastin® (bevacizumabe) é um anticorpo monoclonal inibidor do fator de crescimento endotelial de vasos (VEGF) utilizado "off-label" por meio de administração intravítrea para o tratamento de doenças oculares. A sua aplicação clínica associada ao custo-benefício do medicamento gerou uma demanda para seu fracionamento em frascos de dose única para utilização pela via intraocular. No entanto, a segurança do fracionamento do anticorpo em frascos de dose única ainda é alvo de discussão. Neste trabalho, a estabilidade e a eficácia do Avastin® fracionado em frascos ou ampolas de vidro de dose unitária por farmácias de manipulação do mercado foram avaliadas. Métodos: As técnicas de eletroforese em gel de poliacrilamida (PAGE), cromatografia por exclusão de tamanho (SEC), espalhamento dinâmico da luz (DLS) e turbidimetria foram empregadas para avaliar a formação de agregados de diferentes tamanhos. Alterações na atividade biológica do bevacizumabe foram estudadas utilizando ELISA. Resultados: Amostras referência e do bevacizumabe fracionado apresentaram resultados semelhantes quando analisado por gel de poliacrilamida. Por cromatografia por exclusão de tamanho, um pequeno aumento na quantidade de agregados de alta massa molar seguido de uma redução nos monômeros do bevacizumabe foram observados para as amostras das três farmácias de manipulação quando comparado ao referência. A comparação dos cromatogramas mostrou uma quantidade de redução do monômero inferior a 1% para todas as amostras fracionadas. Por espalhamento dinâmico da luz e turbidimetria, não foram detectados agregados de proteína na faixa de tamanho de micrômetro e nanômetro. No ensaio de eficácia, o bevacizumabe fracionado preservou sua função biológica pois apresentou menos de 3% de perda na capacidade de ligação ao VEGF quando comparado ao referência. Conclusão: Este estudo sugere que o bevacizumabe se mantem estável após fracionamento em ampolas e frascos de vidro de dose unitária pois não foram observadas agregação e/ou fragmentação de proteínas e perda de atividade biológica em quan tidades significativas.

In the final analysis, are we a consumer society or not? Implications for health / Afinal, somos ou não somos uma sociedade de consumo? Consequências para a saúde

In this paper, the question of Brazil's insertion today as a country with the characteristics of modern consumer societies is discussed, focusing on the commercialization of the health sector, the segmentation of the health system and the contradictions of the rights to health care in the social context in question. Some research data on these issues broadcast in the National News Bulletins of Globo TV during the year of 2012 are presented, in which the high technology private hospital as a consumer icon, the underfunding of the public health system and the rejection of a poor and deprived Unified Health System are analyzed.

Discute-se aqui a nossa inserção como país, hoje, nas sociedades de consumo características da modernidade, enfocando a mercantilização na área da saúde, a segmentação do sistema de saúde e as contradições do direito à saúde no contexto social em questão. São apresentados dados de pesquisa sobre o tema no Jornal Nacional da Rede Globo de Televisão, durante o ano de 2012, na qual se analisa o hospital privado de alto padrão tecnológico como ícone de consumo, o subfinanciamento do sistema público de saúde e a rejeição de um Sistema Único de Saúde pobre e carente.

Eliciadores proteicos e glicídicos de Adiantum capillus-veneris L. para fitoalexinas em sorgo Sorghum bicolor (L. ) Moench / Proteinaceous and carbohydrate elicitors from Adiantum capillus-veneris inducing phytoalexins in sorghum

Este trabalho objetivou a purificação parcial, por precipitação com sulfato de amônio (SA) e cromatografia de filtração em gel (CFG), de compostos presentes no decocto de Adiantum capillus-veneris (avenca) eficientes na indução de fitoalexinas em mesocótilos de Sorghum bicolor sorgo. Decocto de A. capillus-veneris a 1% (peso seco/volume) foi precipitado com concentrações de SA variando de 0 a 100% (em intervalos de 20%), e essas frações foram submetidas à CFG. Para o decocto não precipitado foram obtidos nove picos proteicos e um pico glicídico com massas moleculares variando de 0,61 à 0,01 KDa. Para a precipitação fracionada obteve-se: na fração 0-20% dois picos proteicos (menores que 0,01 KDa) e dois glicídicos com concentração de açúcares variando de 4,1 a 17,5 µg mL-1; na fração 20-40% três picos proteicos (111,5 à 0,98 KDa) e cinco glicídicos (11,3 a 73,7 µg de açúcares mL-1); na fração 40-60% dois picos proteicos (111,5 à 0,09 KDa) e dois glicídicos (5,6 a 7, 5 µg de açúcares mL-1); na fração 60-80% seis picos proteicos (menores que 0,02 KDa) e dois glicídicos (16,5 a 51,3 µg de açúcares mL-1); e na fração 80-100% três picos proteicos (menores que 0,09 KDa). Mesocótilos de sorgo foram tratados com as frações provenientes da CFG, além do decocto a 1%, acibenzolar-S-metil (ASM) (125 mg L-1 do i.a. como eliciador de referência) e tampão fosfato de sódio 10 mM pH 6,0. O pico proteico II (0,09 KDa) do decocto não precipitado induziu fitoalexinas, 6,68% superior a ASM. Entre os precipitados, a fração 60-80% de SA induziu 76% mais que ASM. Dessa forma, pôde-se obter frações proteicas e/ou glicídicas indutoras de fitoalexinas em sorgo de maneira superior ao extrato (decocto) do qual é originária, indicando o potencial dessas moléculas para trabalhos futuros sobre indução de resistência.

This study aimed to partially purify the compounds present in decoction of Adiantum capillus-veneris, which are efficient in the induction of phytoalexins in sorghum mesocotyl, by ammonium sulphate (AS) fractionation and gel filtration chromatography (GFC). The decoction of A. capillus-veneris at 1% (weight/volume) was precipitated with AS at the concentration of 0-20%, 20-40%, 40-60%, 60-80% and 80-100%, and these fractions were subjected to GFC. For the decoction not precipitated with AS, nine protein peaks and one carbohydrate peak were obtained with molecular weights ranging from 0.61 to 0.01 KDa. For the AS precipitation, we obtained: for the fraction 0-20%, two protein peaks (0.01 KDa) and two carbohydrate peaks with concentration of sugars ranging from 4.1 to 17.5 µg of sugar mL-1; for the 20-40%, three protein peaks (0.98 to 111.5 KDa) and five carbohydrate peaks (11.3 to 73.7 µg sugar mL-1); for the 40-60%, two protein peaks (0.09 to 111.5 KDa) and two carbohydrate peaks (5.6 to 7.5 µg of sugar mL-1); for the 60-80%, six protein peaks (lower than 0.02 KDa) and two carbohydrate peaks (16.5 to 51.3 µg of sugar mL-1); and for the 80-100%, three protein peaks with molecular weight equivalent to 0.09 KDa. The sorghum mesocotyls were treated with GFC fractions, decoction (1%), acibenzolar-S-methyl (ASM) (125 mg L-1 a.i. as elicitor reference) and sodium phosphate buffer (10 mM, pH 6.0). The protein peak II (0.09 KDa) from the decoction not precipitated was effective in inducing phytoalexin, exceeding in 6.68% the ASM. Among the fractions, the one with 60-80% of AS increased in 76% the induction of phytoalexin compared to ASM. According to the results, we could obtain protein and/or carbohydrate fractions capable of inducing phytoalexins in sorghum better than the decoction from which they are derived from, showing the potential of these molecules for future research studies on the induction of resistance.

Heterogeneity of carboxyl-terminal parathyroid hormone circulating forms in patients with hyperparathyroidism due to end stage renal disease / Heterogeneidade das formas carboxi-terminal circulantes de paratormônio em pacientes com hiperparatiroidismo devido à insuficiência renal crônica terminal

OBJECTIVE: To study carboxyl-terminal (COOH) parathyroid hormone (PTH) circulating forms in patients with hyperparathyroidism due to end stage renal disease (ESRD). METHODS: An immunometric assay that recognizes both intact and COOH PTH forms was developed. The assay, in conjunction with an intact assay, was used to measure PTH in serum samples obtained from 25 patients with hyperparathyroidism due to ESRD. Samples were also submitted to gel filtration chromatography in a Superdex® 30 1.6 x 60 cm column, and the PTH content in the elution tubes, measured using both assays. RESULTS: Values from 39.000 to 232.300 ng/mL (mean ± sd = 101.680 ± 45.330 ng/mL) were found using the COOH assay (PTH 39-84 was used as standard). Values obtained by the intact PTH assay ranged from 318 to 3.307 ng/mL (1.769 ± 693 ng/mL) with a correlation between assays of 0.462 (p = 0.02). The elution profile obtained using the COOH assay showed a preponderance of forms with MW ranging from 8.500 to 4.500 daltons. The profiles obtained from the 25 patients were very similar. CONCLUSIONS: In patients with hyperparathyroidism due to ESRD circulating PTH levels contain a broad range of molecular forms including COOH with MW ranging from 8.500 to 4.500 daltons. These forms are not recognized by the standard intact PTH assays. The correlation of these findings to the clinical aspects of bone disease in ESRD patients remains to be studied.

OBJETIVO: Estudar as formas carboxi-terminal (COOH) circulantes de paratormônio (PTH) em pacientes com hiperparatiroidismo devido à insuficiência renal crônica (IRC) terminal. MÉTODOS: Foi desenvolvido um ensaio imunométrico que reconhece formas intactas e COOH longas de PTH. Esse ensaio foi utilizado, em conjunto com um ensaio para molécula intacta de PTH, em amostras de 25 pacientes com hiperparatiroidismo devido à IRC. As amostras também foram submetidas à cromatografia de gel filtração em coluna de Superdex® 30 de 1,6 x 60 cm, e o conteúdo de PTH nos tubos de eluato foi medido, empregando-se os dois ensaios. RESULTADOS: Valores entre 39.000 e 232.300 ng/mL (média ± dp = 101,680 ± 45,330 ng/mL) foram obtidos usando-se o ensaio COOH (PTH 39-84 foi utilizado como padrão). Com o ensaio para PTH intacto, os valores distribuíram-se entre 318 e 3,307 ng/mL (1,769 ± 693 ng/mL) com correlação entre ambos de 0,462 (p = 0,02). O perfil cromatográfico obtido com o ensaio COOH mostrou predomínio de formas com PM entre 8.500 e 4.500 daltons. Os perfis cromatográficos dos 25 pacientes foram bastante semelhantes. CONCLUSÕES: Em pacientes com hiperparatiroidismo devido à IRC, os níveis circulantes de PTH contêm um espectro de formas moleculares que incluem formas carboxi-terminais, com PM entre 8.500 e 4.500 daltons. Essas formas não são reconhecidas pelos ensaios de rotina utilizados para a medida de PTH intacto. A correlação entre esses achados e os aspectos clínicos da doença óssea em pacientes com IRC necessita de maiores estudos.

Studies on acceleration of Ras cheese ripening by aminopeptidases enzyme from buffaloes' pancreas. I. purification and characterization of buffaloes pancreas aminopeptidas

Aminopeptidaes [EC. 3.4.11.1] was purified from fresh buffaloes pancreas by ammonium sulfate fractionation at 30-40% saturation [w/v], followed by gel filtration chromatography on sephadex G-l 00 column about 9.6 fold with 20% recovery of crude extract. The enzyme had a molecular weight [MW] about 20 kDa by gel filtration chromatography on sephadex G 100 column. The purified enzyme exhibited maximum activity on leucine-p-nitroanilide pH 6.0 and 40°C. The enzyme was strongly activated by 1 mM of Ca [+2], Na[+], respectively, but strongly inhibited by 1 mM of Cu[+]2 and Cd[+]2. The purified aminopeptidase activity was not significantly affected by I mM of EDTA and 1, 10 Phenanthroline

Estudos biofísicos, estruturais e imunológicos de proteínas recombinantes correspondentes a antígenos de superfície de merozoítas de Plasmodium vivax / Studies biophysics, structural and immunological of recombinant proteins corresponding to merozoite surface proteins of Plasmodium vivax antigen

Diversas proteínas de superfície de merozoítas (MSPs) de Plasmodium têm sido consideradas a compor uma vacina contra a malária. Nos últimos anos estudamos diversos aspectos da reposta imune naturalmente adquirida contra proteínas recombinantes baseadas nas MSPs de P. vivax. Estes estudos demonstram que estas proteínas recombinantes mantêm suas funções imunológicas, podendo servir como base para a caracterização de suas estruturas tridimensionais. Com o objetivo de obter informações estruturais sobre as MSPs de P. vivax, 10 proteínas recombinantes, correspondentes à região C-terminal da MSP-1 (MSP119), e diferentes regiões da MSP-3α e MSP-3β foram expressas em Escherichia coli. Os dados estruturais da MSP119 foram obtidos por modelagem molecular com base nas coordenadas cristalográficas da MSP19 de P. cynomolgi. Por outro lado existem poucas informações estruturais sobre as proteínas da família MSP-3 de Plasmodium. A análise da estrutura primária dessas proteínas indica que elas apresentam um domínio central rico em alaninas que estão organizadas como motivos heptads...

Purification and characterization of alkaline proteases from the viscera of mackerel [scumber scumbrus] and macaroni fish [saurus tumbil]

An alkaline protease was extracted from acetone powder prepared from the viscera of Mackerel [Scumber scumbrus] and Macaroni [Saurus tumbil] fish, precipitated from the extract by 40-60% ammonium sulfate, dialyzed and purified by gel exclusion chromatography. The optimal pH and optimal temperature of both enzymes were 8 and 50°C, respectively [using BAPNA as a substrate]. Remarkable stability was observed at pH 6, where more than 75% of the activity of both enzymes remained after 60 min while they retained more than 60% when incubated for the same period at pH 5.0. Furthermore, both enzymes retained more than 50% of their activity after heating at 50°C for 90 min, while, they were completely inactivated when heated at 60°C for 150 min. Gel exclusion chromatography increased the purification to 15.12 and 19.67-fold for Mackerel and Macaroni enzyme extracts, respectively. Only a single protein band with an R[f] value corresponding to a molecular weight of 20,000 Da was observed with the enzyme extracted from Macaroni while two bands with R[f] values close to 20,000 and 28,000 Da were noticed in the Mackerel extract when sodium dodecyl sulphate polyacrylamid gel electrophoresis [SDS-PAGE] was used for molecular weight determination

Efecto agregante plaquetario de 2 citolisinas: sti y stii purificadas de la anémona marina Stichodactyla helianthus / Platelet aggregation effect of 2 cytolysins: sti and stii purified from the sea anemone Stichodactyla helianthus

Las citolisinas Sticholysina I (St I) y Sticholysina II (St II) inducen la agregación plaquetaria en el plasma rico en plaquetas en el rango de concentraciones ensayadas (0,5 a 10 µg/mL). Para ambas citolisinas se obtienen porcentajes de agregación plaquetaria superiores al 90 porciento con menos del 50 porciento de lisis celular. La agregación plaquetaria se mantiene elevada aún cuando la lisis celular disminuye a menos del 20 porciento. El EDTA 2 mM/L y el verapamilo 100 mM/L inhiben significativamente la agregación inducida por StI, lo que evidencia que el calcio extracelular tiene una función importante en este proceso y probablemente esta citolisina tiene una función similar a la de un ionóforo de calcio. Con StII no se obtuvo inhibición significativa de la agregación en presencia de EDTA y verapamilo. La agregación inducida por ambas citolisinas no está influida por el aumento del AMPc intracelular y es independiente de la formación de tromboxano A2 en la plaqueta

Purificación de C1r a partir de la fracción I de Cohn / Purification of C1r starting from Chron's fraction I

Se describe un método de purificación del componente C1r del sistema complemento a partir de la fracción I de Cohn mediante cromatografías secuenciales en Bio Rex 70, DEAE Sephacel y Sephacryl S 200. La presencia de C1r en las fracciones eluidas en las diferentes corridas cromatográficas se detectó por nefelometría. La pureza del C1r obtenido se determinó por inmunoelectroforesis contra un suero anti proteínas séricas humanas y un antisuero específico. A partir del C1r purificado se produjo en conejos un antisuero de buena calidad, útil para la cuantificación del C1r sérico

Immunological assay after vaccination of goats with brucella melitensis rev.I and brucella abortus 45/20 vaccines at Saudi Arabia

The living smooth Brucella melitensis Rev. I vaccine given in the normal dose [7x10[8] organism] to goats 3 to 5 months of age stimulated a marked increase in agglutinin titer. Fractionation of pools of serum by gel filtration by anion exchange chromatography, using diethylaminoethyl [DEAE] cellulose showed that both IgM and IgG agglutinins were present from 12 to 47 days after goats were vaccinated. Only mercaptoethanol [ME]- sensitive agglutinins were detected in most goats 4 months after vaccination, but 2 of 30 goats retained ME- resistant agglutinins for the 13 1/2 - month observation period. Fractionation of serums from goats representative of these 2 types of serologic response indicated that most goats had only IgM agglutinins whereas the 2 given goats had activity in the IgG fraction. Adult goats given Brucella abortus 42/20 adjuvant vaccine and revaccinated 5 months later developed low agglutination titers to smooth antigen, and all became test Positive to the antiglobulin test. Fractionation of serum of pools taken 1 week and 8 months after revaccination indicated that antibody activity was restricted to the IgG fraction. Sera from 5 vaccinated and nonvaccinated goats which were Positive by bacteriological culture examination at necropsy 31 to 47 days after goats were given conjunctival inoculation of virulent B. melitensis had antibody activity in both IgG and IgM fractions Test Positive reaction to the ME, complement-fixation [CF], and antiglobulin [AG] test were restricted to the IgG-containing fractions of serum whereas reactions to the agglutination [STT] and the card tests appeared with either or both fractions. The effect of these findings on the choice of tests for the differentiation of vaccination response is discussed

Gel chromatographic experiments for studying subunit interaction of aldolase and triose-phosphate isomerase

Aldolase and triose-phosphate isomerase [TPI] were prepared and purified from rabbit skeletal muscle by gel chromatographic methods. Molecular weight determination and subunit interaction were demonstrated by gel filtration experiments. Cross-linking of aldolase subunit and TPI subunit with glutaraldehyde was detected from the elution profile of sephadex G/200 column which equilibrated with known proteins. Chemical interaction of the lysyl residue of aldolase with different inhibitors such as acetyl chloride, benzoyl chloride, chloroacetic acid, acetic anhydride, thiourea, thioacetamide and bromo ethyl acetate can prevent the Schiff's base formation and may induce some conformational changes. These conformational changes near catalytic site of aldolase causing inhibition of its catalytic function

Caracterización de formas macroenzimáticas de la fosfatasa alcalina: descripción de un nuevo método / A new method for the characterization of alkaline phosphatase macroenzymes

Se describe un nuevo método para la caracterización de macroenzimas de la fosfatasa alcalina. El método combina una separación previa de las formas isoenzimáticas por gel filtración en capa fina de Sephadex G-200, y el posterior revelado de la correspondiente actividad enzimática in situ. Entre otras ventajas, este sistema no sólo separa adecuadamente las formas macroenzimáticas, sino que mantiene todas las isoenzimas en estado nativo, a diferencia de las técnicas con desnaturalizantes, por lo que es posible su detección y eventual aislamiento. Permite además la resolución simultánea de un gran número de muestras, así como la inclusión de controles de distinto peso molecular (PM) dentro de una misma corrida, lo que facilita en forma significativa la posterior interpretación del perfil obtenido, aportando una gran ventaja con respecto a la técnica de gel filtración en columna. El método propuesto se presenta como una técnica relativamente simple y rápida para el screening de macroenzimas en el laboratorio clínico

Importancia de las isoformas de prolactina sobre la función ovárica en mujeres hiperprolactinémicas / Importance of the prolactin isoforms over the ovarian function in hyperprolactinemic women

El objetivo de este trabajo fue estudiar la importancia de la heterogeneidad molecular de prolactina PRL en muestras de suero de mujeres hiperprolactinémicas sobre la función ovárica. La heterogeneidad molecular de PRL se estudió en muestras de suero en siete pacientes hiperprolactinémicas idiopáticas, mediante cromatografías de filtración en gel (Sephadex G-100). De estas pacientes, cuatro de ellas tenían ciclos menstruales conservados y tres presentaban amenorreas. Los patrones cromatográficos obtenidos de cuatro pacientes con hiperprolactinemia y ciclos menstruales normales mostraron una predominancia de la forma de PRL big-big, de alto peso molecular 79 por ciento Sin embargo, las pacientes con hiperprolactinemia y amenorrea presentaron un patrón cromatográfico con predominancia de PRL little, o de bajo peso molecular 69 por ciento Estos resultados sugieren que la heterogeneidad molecular de PRL estaría asociada con desórdenes en el ciclo menstrual y por ende con la función ovárica, ya que la función ovárica normal en pacientes hiperprolactinémicas parece estar asociada a niveles aumentados de PRL big-big y la función ovárica alterada a niveles altos de PRL little

Purificación rápida de anticuerpos monoclonales empleando un gel TSK -butilo 650 (S) / Rapid purification of monoclonal antibodies using TSK -butil 650 (S) gel

Se evaluó la cromatografía hidrofóbica de alta resolución a nivel analítico como método alternativo en la purificación de anticuerpos monoclonales (AcM) anti interferón alfa 2b humano recombinante. Como soporte fue utilizado un gel TSK-butilo 650(S) de alta rigidez. Se optimizaron las condiciones de corrida para obtener la maxima pureza del AcM anti interferón alfa 2b recombinante CB-IFNA2.1. Resultó necesario modificar el gradiente de elución para purificar otros AcM del mismo isotipo y especificidad, evidenciándose que el gel de butilo muestra una capacidad adicional de diferenciación entre inmunoglobulinas del tipo IgG1. La eliminación de pasos de diálisis y de dasalinización, así como la alta pureza de los anticuerpos obtenidos son las principales características de la purificación utilizando este tipo de matriz

Effectiveness of different methods to eliminate interference by thyroglobulin antibodies in the ELISA for thyroid microsomal autoantibodies.

Thyroid microsomal autoantibodies (TMA) have been mostly detected by means of either immunofluorescence (IF), tanned red cell haemagglutination (TRCH), or radioimmunoassay (RIA) until the recent development of ELISA. False positives in the ELISA for the detection of TMA due to interference by thyroglobulin antibodies (TGA) present in some test sera reacting with thyroglobulin (Tg) present as a contaminant in the thyroid microsomal preparation (TMP) appears to be common. In this study we tried various ways of removing any Tg contaminant in the TMP by further gel filtration, affinity chromatography of the microsomal preparation or preincubation of the test sera with either Tg or Tg-sepharose 4B immunoadsorbent to absorb out TGA present in the sera. Further gel filtration and affinity purification of the TMP failed to totally remove all the contaminating Tg. Preincubation with Tg effectively removed any TGA present in the test sera but resulted in inhibition of the TMA-thyroid microsomal antigen reaction in the test sera including those without TGA. Preincubation with Tg-immunoadsorbent equally effectively absorbed out any TGA present in the test sera but without significant inhibition of the assay reaction in TGA-free sera. The preincubation of the TMP with Tg-immunoadsorbent is an effective way of removing TGA present in sera without inhibiting the test reaction and thus resulting in false negatives especially in low-titre sera as occurs with the presence of free Tg in the test system.

Estudio fitoquímico de semillas de hymenocallis arenicola, amaryllidaceae / Phytochemical study of seeds of hymenicallis arenicola: ameryllidaceae

Se realiza la determinación de alcaloides en semillas de la planta Hymenocallis arenicola, Amaryllidaceae. La extracciòn se desarrolla por el método de Boit y Döpke. Se aislaron 6 componentes por cromatografía de columna sobre silica gel y eluciòn con cloroformo/metanol. La comparaciòn de la información obtenida mediante estudios espectroscópicos con los datos de la literatura, permite identificar estas sustancias como goleptina, tazetina, haemanthidina, haemanthamina, licorina y galanthamina