RESUMO
Reversed-phase and size-exclusion liquid chromatography methods were validated for the assessment of streptokinase. The reversed-phase method was carried out on a Jupiter C4 column (250 mm × 4.6 mm id) maintained at 25°C. The mobile phase consisted of 50 mM sodium sulfate solution pH 7.0 and methanol (90:10, v/v), run isocratically at a flow rate of 0.8 mL/min. The size-exclusion method was carried out on a Protein KW 802.5 column (300 mm × 8.0 mm id), at 25°C. The mobile phase consisted of 40 mM sodium acetate solution pH 7.0, run isocratically at a flow rate of 1.0 mL/min. Retention times were 19.3 min, and 14.1 min, and calibration curves were linear over the concentration range of 0.25-250 µg/mL (25.75-25 750 IU/mL) (r2 = 0.9997) and 5-80 µg/mL (515-8240 IU/mL) (r2 = 0.9996), respectively, for reversed-phase and size exclusion, with detection at 220 and 204 nm. Chromatographic methods were employed in conjunction with the in vitro bioassay for the content/potency assessment of Streptokinase, contributing to improve the quality control and ensure the efficacy of the biotherapeutic.
Assuntos
Bioensaio , Cromatografia Líquida , Ensaios Enzimáticos/métodos , Controle de Qualidade , Estreptoquinase/análise , Cromatografia em Gel , Reprodutibilidade dos Testes , Estreptoquinase/metabolismoRESUMO
Here we demonstrate the usefulness of peptide fractionation by SDS-free polyacrylamide gel electrophoresis and its applicability to proteomics studies. In the absence of SDS, the driving force for the electrophoretic migration toward the anode is supplied by negatively charged acidic amino acid residues and other residues as phosphate, sulfate and sialic acid, while the resulting mobility depends on both the charge and the molecular mass of the peptides. A straightforward method was achieved for SDS-PAGE of proteins, enzyme digestion, peptide transfer and fractionation by SDS-free PAGE, which was named dual-fractionation polyacrylamide gel electrophoresis (DF-PAGE). This method increases the number of identified proteins 2.5-fold with respect to the proteins identified after direct analysis, and more than 80% of assigned peptides were found in unique SDS-free gel slices. A vast majority of identified peptides (93%) have p I values below 7.0, and 7% have p I values between 7.0 and 7.35. Peptide digests that were derived from complex protein mixtures were in consequence simplified as peptides that are positively charged are not recovered in the present conditions. The analysis of a membrane protein extract from Neisseria meningitidis by this approach allowed the identification of 97 proteins, including low-abundance components.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Proteômica/métodos , Caseínas/análise , Caseínas/química , Cromatografia Líquida de Alta Pressão , Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/química , Proteínas de Membrana/análise , Proteínas de Membrana/química , Neisseria meningitidis/química , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Fosfopeptídeos/análise , Fosfopeptídeos/química , Espectrometria de Massas por Ionização por Electrospray , Estreptoquinase/análise , Estreptoquinase/química , Espectrometria de Massas em Tandem , Tripsina/químicaRESUMO
A study was carried out to establish an appropriate method for streptokinase (SK) potency determination (biological assay) in order to fulfil the main function of the Instituto Nacional de Medicamentos respecting products marketed in Argentina. The potency of different commercial samples of SK was determined against the International Standard, and three internationally accepted methods were used for this purpose: fibrin plate, clot lysis and chromogenic method. The analysis of results suggests that the fibrin plate method is the least precise and reproducible. The clot lysis and chromogenic methods demonstrated great precision and reproducibility, giving a correlation coefficient of 0.99. It is concluded that both of these methods are best suited to determine potency of SK commercial products.
Assuntos
Fibrinolíticos/farmacologia , Estreptoquinase/farmacologia , Animais , Bovinos , Compostos Cromogênicos/metabolismo , Estudos de Avaliação como Assunto , Fibrina/metabolismo , Fibrinólise/efeitos dos fármacos , Fibrinolíticos/análise , Fibrinolíticos/normas , Oligopeptídeos/metabolismo , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Estreptoquinase/análise , Estreptoquinase/normasRESUMO
OBJECTIVE: To determine the immune responses to the streptococcal M protein in patients with poststreptococcal acute glomerulonephritis (PSAGN). STUDY DESIGN: The gene coding type 12 M protein of group A streptococcus (M12), a known PSAGN-associated serotype, was cloned and expressed in Escherichia coli to investigate the specific immune responses to the M12 protein in patients with PSAGN. Recombinant M proteins for the variable N-terminal half (AB region) and conserved C-terminal half (C region) were produced separately. IgG titers against each region were measured by enzyme-linked immunosorbent assay in patients with PSAGN (n = 51), uncomplicated streptococcal pharyngitis (n = 26), chronic glomerulonephritis (n = 10), and in healthy control subjects (n = 51). Immunodominant domains within the M protein in PSAGN were further investigated by use of overlapping synthetic peptides. RESULTS: IgG titers against the C region, but not the AB region, were markedly higher in the PSAGN group than in other groups (p < 0.01), and these titers were maintained for at least several months after antistreptolysin O or antistreptokinase levels had returned to normal. Studies with overlapping synthetic peptides demonstrated that increased IgG reactivity was observed against the C repeat blocks. CONCLUSION: IgG titers against the C region are significantly elevated in patients with PSAGN, and it may be a diagnostic marker for PSAGN.