共查询到20条相似文献,搜索用时 24 毫秒
1.
E. Raji Nair Satish Kumar M. V. R. Reddy B. C. Harinath 《Indian journal of clinical biochemistry : IJCB》1998,13(2):98-105
A mycobacterial excretory-secretory protein fraction ESAS-7 purified by 50% ammonium sulphate precipitation followed by SDS-PAGE
fractionation was evaluated by penicillinase enzyme linked immuno-sorbent assay (ELISA) for its sensitivity and specificity
in the diagnosis of pulmonary tuberculosis. At a “cut off” serum dilution of 600, 38 (90%) of 42 sera from bacteriologically
confirmed tuberculosis cases, 15 (100%) of 15 sera from bacteriogically negative but anti tubercular therapy (ATT) responded
cases, 3 (7%) of 43 sera from normal healthy subjects and 4 (8%) of 48 sera from non tuberculous disease control cases gave
positive reaction for tubercular antibody to ESAS-7 antigen fraction containing predominantly 33-kDa protein with a sensitivity
of 90% in bacteriologically confirmed cases and specificity of 92%. Further, this diagnostic assay using the ESAS-7 antigen
is more sensitive requiring as little as one nanogram antigen per test compared to use of 100 nanogram EST-6 antigen reported
earlier. Thus use of ESAS-7 antigen for antibody detection has good diagnostic potential with improved specificity in pulmonary
tuberculosis. 相似文献
2.
BackgroundChinese hamster ovary (CHO) cells are the workhorse for obtaining recombinant proteins. Proteomic studies of these cells intend to understand cell biology and obtain more productive and robust cell lines for therapeutic protein production in the pharmaceutical industry. Because of the great importance of precipitation methods for the processing of samples in proteomics, the acetone, methanol-chloroform (M/C), and trichloroacetic acid (TCA)-acetone protocols were compared for CHO cells in terms of protein recovery, band pattern resolution, and presence on SDS-PAGE.ResultsHigher recovery and similar band profile with cellular homogenates were obtained using acetone precipitation with ultrasonic bath cycles (104.18 ± 2.67%) or NaOH addition (103.12 ± 5.74%), compared to the other two protocols tested. TCA-acetone precipitates were difficult to solubilize, which negatively influenced recovery percentage (77.91 ± 8.79%) and band presence. M/C with ultrasonic homogenization showed an intermediate recovery between the other two protocols (94.22 ± 4.86%) without affecting protein pattern on SDS-PAGE. These precipitation methods affected the recovery of low MW proteins (< 15 kDa).ConclusionsThese results help in the processing of samples of CHO cells for their proteomic study by means of an easily accessible, fast protocol, with an almost complete recovery of cellular proteins and the capture of the original complexity of the cellular composition. Acetone protocol could be incorporated to sample-preparation workflows in a straightforward manner and can probably be applied to other mammalian cell lines as well.How to cite: Pérez-Rodriguez S, Ramírez OT, Trujillo-Roldán MA et al. Comparison of protein precipitation methods for sample preparation prior to proteomic analysis of Chinese hamster ovary cell homogenates. Electron J Biotechnol 2020;48. https://doi.org/10.1016/j.ejbt.2020.09.006. 相似文献
3.
V. M. Baikar M. V. Kamath C. Vishwanathan A. M. Varadkar S. E. Bharmal 《Indian journal of clinical biochemistry : IJCB》2003,18(1):80-86
TMAE-Fractogel 650 (M) (Trimethylaminoethyl) is an ion exchange medium can be used to capture factor VIII (F VIII) directly
from plasma. Previous reports have focused on the use of DEAE-Fractogel 650 (M) (Dimethylaminoethyl) ion exchange medium to
capture F VIII from cryoprecipitate and plasma. Our main objectives were (I) to standardise the purification of FVIII from
human plasma by column chromatographic technique. (II) to study the recovery of FVIII activity in purified fraction at 18–20°C
process condition. (III) to study the effect of virucidal step on recovery of FVIII activity and (IV) to study the effect
of lyophilisation on FVIII activity. In this report, Citrate Phosphate Dextrose (CPD) plasma was batch stirred with dry DEAE-Sephadex
A50, filtered, diluted, loaded on to a column packed with TMAE-Fractogel and chromatographed. Most of the unwanted proteins
flowed through the gel unadsorbed. Bound F VIII was eluted by increasing the ionic strength of the buffer. This purification
step gave an overall 80% recovery from the plasma with a specific activity of 0.97 IU FVIII/mg protein. The purified F VIII
fraction was made virus safe by employing the virucidal technique developed by New York Blood Centre (NYBC). There was 48.43%
loss of FVIII activity in Virus inactivation treatment and the loss of FVIII activity in lyophilisation was 8.45% which is
acceptable. This method of purification gave a higher yield of FVIII than cryoprecipitation, and is a promising alternative
method to cryoprecipitation of F VIII. 相似文献
4.
A. N. Lodam J. Pramanik M. V. R. Reddy P. Narang B. C. Harinath 《Indian journal of clinical biochemistry : IJCB》1997,12(1):71-77
Tuberculosis is emerging as a major public health problem in developing and developed world. Early and precise diagnosis is
of prime importance in successful control of infection. Indirect ELISA with penicillinase as marker was developed using purifiedM. tuberculosis excretory-secretory (EST-DE1) antigen for detecting IgG antibodies in pulmonary tuberculosis. The assay System gave a overall sensitivity of 82% for both
smear positive and smear negative pulmonary tuberculosis cases with a specificity of 84%. The positive and negative predictive
values were 75% and 88% respectivaly. Further studies with EST-DE1 antigen revealed that, it contains two of the active antigen fractions of Mtb EST antigen i.e. Mtb EST-4 (56–68 KDa) and
Mtb EST-6 (37–45 KDa), as demonstrated by inhibition ELISA. Reactivity with monoclonal antibodies HGT 3a showed the presence
of 38 KDa molecule in EST-DE1 antigen. 相似文献
5.
Anubha Paliwal Nivedita Bir Sowmya Raghuraman T. L. P. Reddy P. Usha Sarma 《Indian journal of clinical biochemistry : IJCB》1999,14(2):129-134
Oligonucleotide primers were synthesised based on the gene sequence of an 18 kDa allergen/antigen ofA. fumigatus isolated from a pathogenic strain. Polymerase chain reaction (PCR) was carried out using the forward and reverse primers
and genomic DNA ofA. fumigatus, A. flavus andA. niger as template. This resulted in a PCR product of 480 bp with onlyA. fumigatus. The absence of PCR product inA. flavus andA. niger with the primers of Asp fl facilitated use of these primers for detection ofA. fumigatus in clinical specimens of patients. The results were compared with microscopy, culture and serology. Application of PCR test
to clinical samples of aspergillosis patients is discussed. 相似文献
6.
M. Anindita D. Thamke D. K. Mendiratta B. C. Harinath 《Indian journal of clinical biochemistry : IJCB》2010,25(1):15-19
There is a need for a simple and reliable method to identify Mycobacterium tuberculosis from nontuberculous mycobacteria (NTM).
The utility of mycobacterial ES-31, ES-43, EST-6 or ES-20 antigen as a biomarker for differentiation of Mycobacterium tuberculosis
bacilli from nontuberculous mycobacteria was explored using Fluorescein isothiocyanate conjugated antibodies against these
antigens. Detection of these antigens was done from M.tb H37Ra and H37Rv DSS antigen. The presence of antigen in bacilli using FITC labelled antibody was indicated by green fluorescence on the
cell surface while, its absence by no fluorescence under microscope. In M.tb H37Ra and H37Rv bacilli, fluorescence was observed on addition of FITC labelled anti ES-31 and anti ES-43 antibody; whereas no fluorescence
was observed in case of EST-6 and ES-20 antibody conjugates. However all the antigens were detected in detergent soluble sonicate
antigen of tubercle bacilli on addition of FITC conjugates. Fluorescence was not observed for ES-31, ES-43, EST-6 and ES-20
antigen in any of the tested NTM as well as in Escherichia coli. SEVA TB ES-31 and ES-43 may be used as biomarkers to distinguish
M.tuberculosis bacilli from NTM. 相似文献
7.
S. Bhattacharya K. Sengupta S. Sengupta P. Mukhopadhyay A. Debnath P. Das 《Indian journal of clinical biochemistry : IJCB》1998,13(1):33-35
Summary Amoebiasis, caused by an enteric protozoanEntamoeba histolytica, is one of the major parasitic diseases of mankind. Current estimate suggests that the parasite infects about 10% of the
world population at any given time. There is an urgent need to characterize the antigenic molecules ofE. histolytica, and find out antigens which have both immunodiagnostic and prophylactic potential against amoebiasis. The results of somatic
antigen analysis, using sera from immune or infected individuals, indicated that the wholeE. histolytica trophozoites, are highly complex and heterogeneous in nature and both immunodiagnostic and immuno prophylaxis activity remain
mainly in a surface associated 29 kDa glycoprotein ofE. histolytica. Future studies at molecular level particularly, genes responsible for expression of this protein, their homology with other
proteins and structure analysis will give better understanding about this polypeptide. Studies on excretory secretory antigens,
clearly demonstrated thatE. histolytica like many organisms, also liberates certain antigenic moieties into the culture medium during in vitro cultivation and this
antigen has similar diagnostic values like the conventional somatic antigens. It is important that the ESA should be prepared
from the supernatant after collecting the cell and use for immunodiagnosis of amoebiasis. This is an additional source of
antigen which will help in carrying out more tests using the same reagents. Further studies are needed to clarify the location
of these molecules on the parasite, along with detailed biochemical and immunological characterization and their relation
with the pathogenesis.
This was presented at the symposium on “Recent Trends in Tropical Disease Research”, held at Sevagram on 5-6 September, 1997. 相似文献
8.
9.
BackgroundThe determination of kinetic parameters and the development of mathematical models are of great interest to predict the growth of microalgae, the consumption of substrate and the design of photobioreactors focused on CO2 capture. However, most of the models in the literature have been developed for CO2 concentrations below 10%.ResultsA nonaxenic microalgal consortium was isolated from landfill leachate in order to study its kinetic behavior using a dynamic model. The model considered the CO2 mass transfer from the gas phase to the liquid phase and the effect of light intensity, assimilated nitrogen concentration, ammonium concentration and nitrate concentration. The proposed mathematical model was adjusted with 13 kinetic parameters and validated with a good fit obtained between experimental and simulated data.ConclusionsGood results were obtained, demonstrating the robustness of the proposed model. The assumption in the model of DIC inhibition in the ammonium and nitrate uptakes was correct, so this aspect should be considered when evaluating the kinetics with microalgae with high inlet CO2 concentrations.How to cite: Saldarriaga L F, Almenglo F, Ramírez M, et al. Kinetic characterization and modeling of a microalgae consortium isolated from landfill leachate under a high CO2 concentration in a bubble column photobioreactor. Electron J Biotechnol 2020;44. https://doi.org/10.1016/j.ejbt.2020.01.006. 相似文献
10.
Bonny Bhunia R. S. Alli M. V. R. Reddy B. C. Harinath 《Indian journal of clinical biochemistry : IJCB》1999,14(2):143-148
Brugia malayi microfilarial excretory-secretory (mf ES) antigens obtained byin vitro maintenance of mf are important tools in the immunodiagnosis of bancroftian filariasis. To increase the yield of mf ES products,
the effect of nutritional supplements on the culture medium (RPMI 1640) and the maintenance temperature were studied. Supplementation
of RPMI 1640 medium with organic acids and sugars of Grace's insect culture medium forin vitro maintenance of 10 lakhs of mf in 40 ml medium increased the yield of mf ES antigen from 152 μg to 364 μg of mean protein,
content and the mean antigen titre from 200 to 400. Supplementation with phenyl methyl sulphonyl fluoride (PMSF) and shift
in the culture temperature resulted in a further increase in the yield of mf ES antigen to 502 μg of mean total protein with
an antigen titre of 800. The modification resulted in a net increase of 3 fold in the protein content and 4 fold in the antigen
titre of ES products. The above modifications in thein vitro maintenance of mf did not affect the diagnostic quality of mf ES antigen, which gave a sensitivity and specificity of 80%
and 90% respectively in detection of filarial IgG antibodies inWuchereria bancrofti infected cases. 相似文献
11.
We have designed, built, and evaluated a microfluidic device that uses deterministic lateral displacement for size-based separation. The device achieves almost 100% purity and recovery in continuously sorting two, four, and six micrometer microspheres. We have applied this highly efficient device to the purification of fungal (Aspergillus) spores that are spherical (∼4 μm diameter) with a narrow size distribution. Such separation directly from culture using unfiltered A. niger suspensions is difficult due to a high level of debris. The device produces a two to three increase in the ratio of spores to debris as measured by light scatter in a flow cytometer. The procedure is feasible at densities up to 4.4×106 spores∕ml. This is one of the first studies to apply microfluidic techniques to spore separations and has demonstrated that a passive separation system could significantly reduce the amount of debris in a suspension of fungal spores with virtually no loss of spore material. 相似文献
12.
P. Sharma M. Bose Isa Mohd S. Bagdi H. G. Raj 《Indian journal of clinical biochemistry : IJCB》2000,15(2):83-87
Genomic DNA from a clinical isolate ofMycobacterium avium-intracellulare complex was purified and cloned in PBR 322 at the tetracycline resistance site using Bam HI restriction enzyme. A 16 kb cloned
fragment was purified, radiolabeled and used as a probe. Genomic DNA isolated from nineteen MAC strains, threeM. tuberculosis strains and oneM. kansasii strain were digested with Eco RI restriction enzyme, Southern blotted and hybridized with the 16 kb cloned and labeled fragment.
Twelve MAC strains showed positive hybridization although five strains gave faint signals. Positive hybridization was noted
in two out of the threeM. tuberculosis strains, possibly due to shared DNA homology. No signal was received from the singleM. kansasii strain used in this study. 相似文献
13.
BackgroundBiomineralization is a significant process performed by living organisms in which minerals are produced through the hardening of biological tissues. Herein, we focus on calcium carbonate precipitation, as part of biomineralization, to be used in applications for environmental protection, material technology, and other fields. A strain GM-1, Microbacterium sp. GM-1, isolated from active sludge, was investigated for its ability to produce urease and induce calcium carbonate precipitation in a metabolic process.ResultsIt was discovered that Microbacterium sp. GM-1 resisted high concentrations of urea up to 60 g/L. In order to optimize the calcification process of Microbacterium sp. GM-1, the concentrations of Ni2 + and urea, pH value, and culture time were analyzed through orthogonal tests. The favored calcite precipitation culture conditions were as follows: the concentration of Ni2 + and urea were 50 μM and 60 g/L, respectively, pH of 10, and culture time of 96 h. Using X-ray diffraction analysis, the calcium carbonate polymorphs produced by Microbacterium sp. GM-1 were proven to be mainly calcite.ConclusionsThe results of this research provide evidence that Microbacterium sp. GM-1 can biologically induce calcification and suggest that strain GM-1 may play a potential role in the synthesis of new biominerals and in bioremediation or biorecovery. 相似文献
14.
Swati Banerjee Sonika Gupta Niraj Shende Satish Kumar Bhaskar C. Harinath 《Indian journal of clinical biochemistry : IJCB》2003,18(2):48-53
Serodiagnosis by ELISA has been widely explored over the years, in the diagnosis of tuberculosis. Two ELISA systems were evaluated
for detection of mycobacterial antibodies in pulmonary and extra pulmonary tuberculosis. The two test assays explored were
ERBA LISA (TB IgG) test (Anda Biologicals) which uses A60 antigen complex found in the cytosol of typical and atypical mycobacteria,
and SEVA TB (IgG) ELISA, which uses a 31 kDa, glycoprotein antigen purified fromM. tb H37Ra culture filtrate. Sera from 98 proven tuberculosis [pulmonary TB (48), tuberculous lymphadenopathy (30), tuberculous meningitis
(15) & genitourinary TB (5)] were studied along with 32 healthy controls. The overall positivity obtained using ERBA LISA
(TB IgG) test and SEVA TB (IgG) ELISA test was 72.9% and 91.6% in pulmonary tuberculosis, 43.3% and 76.6% in tuberculous lymphadenopathy
respectively. The sensitivity of ERBA LISA test in tuberculous meningitis and genito-urinary TB was significantly low (26.6%
& 40% respectively) compared to sensitivity obtained using SEVA TB ELISA (86.6% & 60% respectively) with overall specificity
of 60% and 87.5%. Thus SEVA TB IgG ELISA test was found to be more sensitive than ERBA LISA in detecting IgG antibodies in
tuberculous sera, in particular in extra pulmonary tuberculosis cases. 相似文献
15.
16.
D. Saravanakumar N. Elangeswaran S. Senthilkumar G. Vanaja S. Kamakshiammal C. Chandrasekar C. N. Deivanayagam Manjula Sritharan V. Sritharan 《Indian journal of clinical biochemistry : IJCB》2000,15(2):94-103
We have isolated and identified the biotype of environmental mycobacteria from the expectorate of leprosy patients, their
contacts, their drinking water supply and also from the sputa samples of tuberculosis patients. 78% of the isolates from lepromatous
leprosy patients and their contacts wereMycobacterium fortuitum- chelonae complex (MFC), 9%Mycobacterium avium complex (MAC), 9%Mycobacterium scrofulaceum and 4% wereMycobacterium smegmatis. Among the isolates from tuberculosis patients 63% belonged toM. fortuitum- chelonae complex, 19% toM. avium complex, 12% toMycobacterium Kansasii and 6% toM. smegmatis. All the isolates were multi-drug resistant when tested for sensitivity total of 21 drugs. TheMycobacterium fortuitum-chelonae complex organisms from leprosy contacts were more sensitive to rifampicin than those isolated from lepromatous leprosy and
tuberculosis patients. Among 23 isolates from leprosy patients one isolate was resistant to 20 drugs, one isolate to 17 drugs
and another isolate was resistant to 13 drugs. Among the 18 isolates from drinking water supply six showed resistance to more
than 12 drugs. Polymerase Chain Reaction (PCR) and subsequent hybridisation with specific probes confirmed all the isolated
strains as nontuberculous mycobacteria (Using genus primers and probe sensitivity 100%) and none asM. tuberculosis, suggesting that PCR could be used to rapidly identify mycobacteria at the genus level and to rule out tuberculosis in leprosy
patients at an early stage to decide on appropriate course of therapy. 相似文献
17.
Ajay Kumar T. A. Venkita Subramanian Usha P. Sharma 《Indian journal of clinical biochemistry : IJCB》1992,7(2):165-168
Protein fraction termed P2 was obtained from sonicates of Mycobacterium smegmatis, subjected to ammonium sulphate precipitation. P2 fraction was further fractionated into 4 fractions (PeakI-IV) by DEAE cellulose ion-exchange chromatography. ELISA was performed
on the sera of 104 tuberculous cases and 62 controls, using P2 fractions Peak I as test antigens. Though the mean ELISA values in tuberculous cases were higher than the controls, no statistically
significant difference was found between the two. Antigens P2, Peak I and IV were tested on Western blots with pooled sera from tuberculous cases as well as controls. P2 fraction, Peak-I and IV were separated on PAGE-SDS, electroblotted onto nitrocellulose Sheets and the blots were subjected
to ELISA. Peak IV appeared as a single band (M.W. 55,000). P2 fraction exhibited some discriminatory bands on development of blots. 相似文献
18.
BackgroundThe increasing rate of breast cancer globally requires extraordinary efforts to discover new effective sources of chemotherapy with fewer side effects. Glutaminase-free l-asparaginase is a vital chemotherapeutic agent for various tumor malignancies. Microorganisms from extreme sources, such as marine bacteria, might have high l-asparaginase productivity and efficiency with exceptional antitumor action toward breast cancer cell lines.Resultsl-Asparaginase-producing bacteria, Bacillus velezensis isolated from marine sediments, were identified by 16S rRNA sequencing. l-Asparaginase production by immobilized cells was 61.04% higher than that by free cells fermentation. The significant productivity of enzyme occurred at 72 h, pH 6.5, 37°C, 100 rpm. Optimum carbon and nitrogen sources for enzyme production were glucose and NH4Cl, respectively. l-Asparaginase was free from glutaminase activity, which was crucial medically in terms of their severe side effects. The molecular weight of the purified enzyme is 39.7 KDa by SDS-PAGE analysis and was ideally active at pH 7.5 and 37°C. Notwithstanding, the highest stability of the enzyme was found at pH 8.5 and 70°C for 1 h. The enzyme kinetic parameters displayed Vmax at 41.49 μmol/mL/min and a Km of 3.6 × 10−5 M, which serve as a proof of the affinity to its substrate. The anticancer activity of the enzyme against breast adenocarcinoma cell lines demonstrated significant activity toward MDA-MB-231 cells when compared with MCF-7 cells with IC50 values of 12.6 ± 1.2 μg/mL and 17.3 ± 2.8 μg/mL, respectively.ConclusionThis study provides the first potential of glutaminase-free l-asparaginase production from the marine bacterium Bacillus velezensis as a prospect anticancer pharmaceutical agent for two different breast cancer cell lines.How to cite: Mostafa Y, Alrumman S, Alamri S, et al. Enhanced production of glutaminase-free L-asparaginase by marine Bacillus velezensis and cytotoxic activity against breast cancer cell lines. Electron J Biotechnol 2019;42. https://doi.org/10.1016/j.ejbt.2019.10.001. 相似文献
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银杏叶提取物(Gink Biloba Extract,GBE)是银杏干燥叶通过中药提取方法制成的提取物。文章分析了历年来相关GBE的提取和分离纯化方法,如常规提取法的水提取法,有机溶剂萃取法,树脂吸附分离法;新型提取法的超临界CO2萃取法,超声波提取法,高速逆流色谱法等以及GBE应用于牙膏的相关研究进展情况。 相似文献