Showing 7 results for Chromatography
H Tajerzadeh, S Sadray,
Volume 13, Issue 3 (11-1999)
Abstract
A simple, rapid and sensitive HPLC method for the determination of theophylline
(T) in human serum has been developed. An isocratic system consisting of a /-l
Bondapak C18 column, mobile phase of methanol, phosphate buffer (22:78, pH=4.5),
and a flow rate of 1.4 mL/min was used. The eluent was detected by UV at 275 nm
at room temperature. 8-Chlorotheophylline (8-CT) was used as an internal standard.
The serum samples deproteinated by methanol containing 8-CT and the supernatant
was injected into the HPLC system. The retention times of 5.7 and 8.1 min were
found for T and 8-CT, respectively. The linearity was checked in the range of 0.2-
30 µg/mL. Relative standard deviation for both inter-day and intra-day precision
analysis was less than 5%. No interference was observed from endogenous serum
components. Specificity was shown against some commonly co-administered drugs.
Simple and fast sample preparation, small sample volume (250 µL), precision,
reproducibility, specificity, sensitivity and high percentage recovery (98 %) make the
method to be practically useful for T monitoring in asthmatic patients.
Durdi Qujeq, Q Usefnia Pasha,
Volume 14, Issue 3 (11-2000)
Abstract
We have developed a simple and precise paper chromatographic method for
the determination of 4-hydroxy-3-methoxymandelic acid (VMA) in urine. Concentrations
of VMA in patients with neuroblastoma were increased in comparison
to controls. The linearity was excellent in the concentration range tested. The
within-assay coefficient of variation for control and patient urine was less than
2.2%. The recovery was in the range of 97.9-99.4%. Results from testing urine
samples of controls and patients with neuroblastoma suggest that this method is a
reliable and convenient system for quantification of VMA in urine and can be
used in the mass screening of neuroblastoma in infants. Sample preparation requires
minimal time and the entire procedure is completed within 5 h.
H Goudarzi, B Kazemi, F Falah, G Eslami, M Ghazi, F Doustdar,
Volume 17, Issue 2 (8-2003)
Abstract
Tuberculosis remains as an important socioeconomical and medical problem
throughout the world and especially in Iran. Early and timely diagnosis of
pulmonary and extrapulmonary tuberculosis is vital to initiate prompt treatment.
Current diagnostic methods are either slow or lack enough sensitivity or specificity.
Several mycobacterial antigens are involved in the complex interaction with
the immune system of the host. Their identification is important for both diagnosis
and protection against mycobacteria. Antigen 60 (A60) is a thermostable antigen
found in the cytosol of M. bovis and M. tuberculosis. An ELISA test using
A60 is designed for diagnosis of tuberculosis with satisfactory results. In previous
studies, A60 has also showed a protective effect against experimental infections
and useful immunotherapeutic effects in promotion of cancer development.
In the present work we tried to purify A60 from the cytoplasm of BCG. A60
was purified by exclusion gel chromatography using sepharose 4B. A60 was
recognized by bidimensional immunoelectrophoresis with anti-BCG and anti-A60
antiserum, where it appears as the less mobile component. In agarose electrophoresis,
A60 showed only one band but in immunodiffusion it showed two
immunoprecipitinogen lines with anti-BCG anti-serum. In analyzing with dot blotting,
both cytoplasm and cell wall of BCG showed positive reaction with antiA60
anti-serum. When A60 was fractionated by SDS-PAGE and analyzed by:
western blot using anti-A60 antibody, 65,46, 40, 38 and 35 KDa protein fractions'
were identified.
It is concluded that A60 is a macromolecular antigen of BCG with a molecular
weight of 106_107 Da and is a lipoprotein-polysaccharide complex which contains
several proteins. A60 is present in both cytoplasm and cell wall of BCG and
can easily be purified from BCG vaccine using exclusion chromatography by
sepharose 4B, to be used for designing diagnostic tests for TB.
Kamran Mousavi Hosseini, Mojgan Pourmokhtar, Rassoul Dinar Vand, Houri Rezvan, Mohammad Ali Jalili,
Volume 17, Issue 4 (2-2004)
Abstract
As IgM and IgA-enriched preparations are needed to complete the immunotherapeutic
spectrum, a simple procedure is described for the preparation of IgM and IgA enriched
immunoglobulins. Fraction III which was prepared by cold ethanol fractionation
was treated by octanoic acid followed by ethanol precipitation and ion-exchange
chromatography using Sephadex DEAE A-50 and 0.1 M tris-D.35M NaCI buffer, pH
8.1, resulting in recovery of 85 % IgM, 84% IgA and 33 % IgG. The comparison of our
results with immunoglobulins' percentage in plasma indicates that IgM and IgA -enrichment
was obtained by three times.
Kamran Mousavi Hosseini, Saleh Nasiri,
Volume 29, Issue 1 (1-2015)
Abstract
Background: Factor VII concentrates are used in patients with congenital or acquired factor VII deficiency or treatment of hemophilia patients with inhibitors. In this research, immunoaffinity chromatography was used to purify factor VII from prothrombin complex (Prothrombin-Proconvertin-Stuart Factor-Antihemophilic Factor B or PPSB) which contains coagulation factors II, VII, IX and X. The aim of this study was to improve purity, safety and tolerability as a highly purified factor VII concentrate.
Methods : PPSB was prepared using DEAE-Sephadex and was used as the starting material for purification of coagulation factor VII. Prothrombin complex was treated by solvent/detergent at 24°C for 6 h with constant stirring. The mixture of PPSB in the PBS buffer was filtered and then chromatographed using CNBr-activated Sepharose 4B coupled with specific antibody. Factors II, IX, VII, X and VIIa were assayed on the fractions. Fractions of 48-50 were pooled and lyophilized as a factor VII concentrate. Agarose gel electrophoresis was performed and Tween 80 was measured in the factor VII concentrate.
Results : Specific activity of factor VII concentrate increased from 0.16 to 55.6 with a purification-fold of 347.5 and the amount of activated factor VII (FVIIa) was found higher than PPSB (4.4-fold). Results of electrophoresis on agarose gel indicated higher purity of Factor VII compared to PPSB these finding revealed that factor VII migrated as alpha-2 proteins. In order to improve viral safety, solvent-detergent treatment was applied prior to further purification and nearly complete elimination of tween 80 (2 μg/ml).
Conclusion : It was concluded that immuonoaffinity chromatography using CNBr-activated Sepharose 4B can be a suitable choice for large-scale production of factor VII concentrate with higher purity, safety and activated factor VII.
Raheleh Rafiei Sefiddashti, Seyedeh Maryam Sharafi, Soltan Ahmad Ebrahimi, Lame Akhlaghi, Ali Moosavi, Abbasali Eskandarian, Alireza Hejrati, Hossein Yousofi Darani,
Volume 31, Issue 1 (1-2017)
Abstract
Background: Hydatid disease is characterized by long-term growth of hydatid cysts in the human. The glycan antigens have an
important role in the immunology of hydatid cyst. In this study immunological reaction of host sera to different glycan antigens of the
cyst, has been investigated.
Methods: The antibody responses were tested to glycoprotein and glycolipid of the laminated layer (LL), cyst fluid (CF) and protoscolex
(PS) antigens of E. Granulosus using ELISA and western immunoblotting tests. Thin-layer chromatography and ß-elimination
were used for glycan purification.
Results: Both hydatid cyst and normal human sera reacted with hydatid cyst fluid, protoscolices, laminated layer, glycoprotein and
glycolipid antigens. The most antigen-antibody reaction was related to CF and PS antigens, and LL antigens had the minimal reaction
with the sera. Thin layer chromatography (TLC) of the antigens showed presence of many glycan bands in the laminated layer.
Conclusion: The parasite may elaborate different glycan antigens in LL to evade host immune response.
Samaneh Gholami, Koroush Goodarzvand Chegini, Nematollah Gheibi, Kobra Mokhtarian, Mohsen Mohamadi, Reza Falak,
Volume 31, Issue 1 (1-2017)
Abstract
Background: Betatrophin, a novel secretory protein from liver and fatty tissues, is believed to be involved in lipid and glucose metabolism. However, its precise physiological role remains unclear. Here, we report the cloning, expression, and purification steps of mouse betatrophin in a prokaryotic system, followed by its structural analysis.
Methods: Specific cloning primers were used to amplify the coding sequence of mouse liver betatrophin. The product was cloned into pET28 and expressed in E.coli BL21 (DE3) cells. The suitability of the refolding procedure was assessed by determining secondary structures of the initial and refolded proteins using circular dichroism spectroscopy.
Results: The polymerase chain reaction resulted in a 549 bp nucleotide sequence, encoding a 183 amino acid polypeptide, with an apparent molecular weight of 21 kDa, which was expressed in an inclusion body. Following an optimization and refolding procedure, the recombinant protein was purified by anion exchange and metal affinity chromatography. CD spectra revealed that the refolded protein has suitable configuration.
Conclusion: We believe that the produced betatrophin is suitable for further biochemical studies on glucose and lipid metabolism.
