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.

Two-step Tandem Chromatography Purification of Anti-human CD80 Monoclonal Antibody 4E5 from Mouse Ascites / 中国生物工程杂志

A two-step method for the purification of blocking-type anti-human CD80 monoclonal antibody 4E5 from mouse ascites was developed using anion exchange and gel filtration in combination. The ascites was first purified by anion exchange after centrifugation and filtration. The experimental parameters of sample loading and elution were optimized. The optimized loading condition was pH 8.0,50 mmol/L Tris-HCl and satisfactory results were obtained using a 0~0.5mol/L NaCl step elution. The fraction containing the protein of interest was directly loaded on gel filtration column and eluted using a 20 mmol/L phosphate buffer at pH 7.2. The purity of the obtained monoclonal antibody was up to 95% with a recovery of 61%. The purity of mAb could efficiently inhibit the growth of Daudi cells. The amplification of the method was also studied using a Bio-Scale Q5 column and the result was satisfied.

Isolation, purification and content determination of melittin from bee venom / 第二军医大学学报

Objective:To isolate and purify melittin from bee venom and to establish an SDS PAGE and HPLC method for the determination of melittin content.Methods:Melittin was isolated and purified from bee venom by gel filtration on Sephadex G 25,Sephadex G 50 and Sephadex G 75.Homogenicity and molecular weight of melittin were determined by SDS PAGE and HPLC.Results:The sample was a single peptide chain with a molecular mass of about 3 000 dalton as revealed by SDS PAGE.The average recovery was 95.97% and RSD was 1.07%.Conclusion:Melittin prepared by a combination of gel filtration has high purity.The method for the determination of melittin with SDS PAGE and HPLC is accurate,reliable and simple. [