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کاربرد نوع شرط:
- جایگاه : پژوهشی
- مجله: Journal of Applied Biotechnology Reports
- نوع مقاله: Journal Article
- کلمات کلیدی: Enzyme-Linked Immunosorbent Assay (ELISA),Glomerular filtration rate (GFR),Creatinine,Cystatin C
- چکیده:
- چکیده انگلیسی: Introduction: Chronic kidney disease is a worldwide health problem and Glomerular filtration rate (GFR) is the most frequently used criteria in the assessment of rental function. Cystatin C as a member of type 2 cystatin superfamily and cysteine-protease inhibitor is found in high concentrations in all biological fluids. This study is aimed to study cystatin C as a potent biomarker for clinical measurement of renal disorders and other diseases.
Materials and Methods: In this study, the human cystatin C construct was analyzed by bioinformatics software. It was cloned and expressed to produce an appropriate antigen for anti-cystatin C (anti-Cys C) obtained from mice Balb/C as a crucial point in the improvement of an enzyme-linked immunosorbent assay (ELISA) method. Serum samples were given from 32 hospitalized patients with renal failure and cardiovascular disease and non-hospitalized patients were tested by ELISA method using anti-Cys C obtained from mice Balb/C.
Results: Our findings indicated 0.36-2.4 mg/L as the best conclusion for antigen cystatin C in patients’ sera and 1/50 dilution for anti-Cys C obtained from mice Balb/C and showed a relationship between patients with high creatinine and high concentration of cystatin C. In case of five cardiovascular disease patients with normal upper limit of Creatinine we obtained cystatin C lower than kidney failure and raising of cystatin C in 6 patients with increased TSH were seen.
Conclusions: Polyclonal anti-Cys C antibodies were obtained through the immunization of Balb/C mice can be employed as an anti-Cys C in ELISA for diagnosis of some renal dysfunction.- انتشار مقاله: 20-08-1396
- نویسندگان: Zahra Abdollah,Samaneh Khodi,Elaheh Gheybi,Jafar Amani,Ali Karami
- مشاهده
- جایگاه : پژوهشی
- مجله: Journal of Applied Biotechnology Reports
- نوع مقاله: Journal Article
- کلمات کلیدی: PCR-ELISA,Molecular detection,New Methods
- چکیده:
- چکیده انگلیسی: Due to the spread of infectious diseases, the existence of a rapid and sensitive detection method is necessary today. Polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA) is a simple manner for detection of microorganism. For example, bacteria, viruses, fungi and others based on nucleic acid sequence. A large number of samples can be screened by this technique simultaneously, so it is not time consuming and is a quick manner. The high sensitivity and specificity of PCR-ELISA make it a powerful technique by simple laboratory facilities. As a result it can be an excellent substituted manner for analysis and detectionin different various fields.
- انتشار مقاله: 21-03-1396
- نویسندگان: Fatemeh Tayebeh,Shahram Nazarian,Seyed Ali Mirhosseini,Jafar Amani
- مشاهده
- جایگاه : پژوهشی
- مجله: Journal of Applied Biotechnology Reports
- نوع مقاله: Journal Article
- کلمات کلیدی: Klebsiella pneumoniae,16S rDNA,PCR-ELISA,Diagnostic Method
- چکیده:
- چکیده انگلیسی: Klebsiella pneumoniae is the most important infectious bacteria in Enterobacteriaceae family and the most common bacteria causing Urinary Tract Infection (UTI) after Escherichia coli. Therefore, accurate and rapid identification of this bacterium in hospital infection is very important.In this study, PCR-ELISA method was used for detecting Klebsiella pneumonia clinical strains. For this purpose, 16S rDNA gene based specific primers were designed and the DIG-labeled PCR products were bound to streptoavidin-coated wells of a microtiter plate and detected by anti-DIG–peroxidase conjugate. Biotin-labeled DNA probe specific for 16S rDNA gene was used in PCR-ELISA.Sensitivity and specificity of PCR-ELISA method were determined by using Enterobacteria strains. 16S rDNA of Klebsiella pneumoniae was amplified using gene specific primers resulted in a fragment of the 260 bp. The results of PCR-ELISA showed that this technique does not cross-react with the bacteria in their families as well as the sensitivity of 6.0 ng were evaluated.PCR-ELISA is known as an accurate and rapid method for detection of the infectious agents and therefore can be used as a suitable substitute for all the above aspects because it is quite a sensitive, specific, and rapid method for detection of the Klebsiella pneumoniae strains.
- انتشار مقاله: 26-06-1395
- نویسندگان: Fatemeh Tayebeh,Jafar Amani,Shahram Nazarian,Mehdi Moradyar,Seyed Ali Mirhosseini
- مشاهده
- جایگاه : پژوهشی
- مجله: Journal of Applied Biotechnology Reports
- نوع مقاله: Journal Article
- کلمات کلیدی: Cytomegalovirus,Polytope DNA Vaccine,In silico Study,Epitope Maping
- چکیده:
- چکیده انگلیسی: Human cytomegalovirus is one of the most common pathogenic viruses all over the world. In congenital infection leads to neurologic severe disorders and even death of fetus and in individuals with immunosuppression may also cause severe clinical symptoms. Multiple evidence indicate that among several strategies, epitope-based vaccine (EVs) that can induce both humoral and cellular immunity responses, are the most important and have numerous potential profits. In this study, we select the viral surface glycoprotein B and phosphoprotein 65 and 150 with the highest antigenic and immunogenic properties, that have the most important role in induce cellular and humoral immune responses. Bioinformatics tools, as a standard and developed approaches use for epitope mapping. Epitope discovery greatly accelerate by in silico prediction methods with in vitro and in vivo verification. Bioinformatics methods and epitopes identification algorithms were used in order to selection of cytomegalovirus immunodominant epitopes, detection of each epitope antigenicity and design chimeric gene construct. The chimeric protein showed high antigenicity in vaxiJen analysis. Also further immunoinformatic analyses in order to predict the discontinuous and continuous B and T cell epitopes and MHC binding peptides affinity were used. The study results show that protein structures were suitable. Therefore it can be expected that construct is proper subject for practical experiments and stimulus for humoral and cellular immune responses.
- انتشار مقاله: 24-06-1393
- نویسندگان: Elaheh Sabbaghian,Fatemeh Roodbari,Alireza Rafiei,Jafar Amani
- مشاهده
- جایگاه : پژوهشی
- مجله: Journal of Applied Biotechnology Reports
- نوع مقاله: Journal Article
- کلمات کلیدی: Nanoparticles,Vaccine Delivery,Nanovaccines,Vaccine development
- چکیده:
- چکیده انگلیسی: Vaccination has greatly improved human health. Despite of all improvements in this field, there is not an efficient vaccine for many diseases, and of the available ones, some could not produce a long-term immunity. Recently, there have been many researches on the applicability of nanostructures as an efficient system for vaccine delivery, and the initial results have been promising. Their potential adjuvanticity, capability of the stimulation of both humoral and cellular immunity responses, more stability in environmental conditions, possible targeted vaccine delivery, the need for low quantity of proteins (in the case of subunit vaccines), etc., are of the main reasons that this area has gained many interests. Here, we try to review the main nanostructures that could be act as a delivery vehicle in vaccine delivery.
- انتشار مقاله: 24-06-1393
- نویسندگان: Abbas Hajizade,Firouz Ebrahimi,Ali-Hatef Salmanian,Ayyoob Arpanaei,Jafar Amani
- مشاهده
- جایگاه : پژوهشی
- مجله: Journal of Applied Biotechnology Reports
- نوع مقاله: Journal Article
- کلمات کلیدی: Pyrroline-5-Carboxylate Synthase,Amino Acid Substitutions,Tertiary Structure Modeling
- چکیده:
- چکیده انگلیسی: Mammalian ∆-(1)-Pyrroline-5-carboxylate synthase (P5CS) enzyme catalyzes the coupled phosphorylation and reduction-conversion of glutamate to ∆-(1)-pyrroline-5-carboxylate (P5C), a critical step in the proline, ornithine, citrulline and arginine biosynthesis. In plants and mammals, P5CS consists of two separate enzymatic domains: N-terminal γ-glutamyl kinase (γ-GK) and C-terminal γ-glutamyl phosphate reductase (γ–GPR). Hyperammonemia has been reported as a new inborn disorder, with a range of clinical symptoms which is associated with a reduced synthesis of proline, ornithine, citruline and arginine. A missense mutation, R84Q, which alters the conserved residue in γ-GK domain, is responsible for this disorder. In this study using in-silico approaches as a new bioinformatics method, sequence analysis was performed and the tertiary structure of γ-GK domain of human P5CS, which includes the R84Q missense mutation, was predicted and the mutation effects on structural and functional features of P5CS enzyme were analyzed. Our analysis showed that this substitution has an affect on the molecular surface accessibility and total energy of the modeled structure. We conclude that this mutation results in a reduced activity of P5CS enzyme and an impaired synthesis of these amino acids.
- انتشار مقاله: 26-11-1392
- نویسندگان: Maryam Zare,Faranak Hadi,Zarrin Minuchehr,Jafar Amani,Ali Hatef Salmanian
- مشاهده
- جایگاه : پژوهشی
- مجله: Iranian Journal of Pathology
- نوع مقاله: Journal Article
- کلمات کلیدی: Cancer therapy,In silico modeling,diphtheria toxin,shiga like toxin part B,bioinformatics tools
- چکیده:
- چکیده انگلیسی: Background & Objective:
A main contest in chemotherapy is to obtain regulator above the biodistribution of cytotoxic drugs. The utmost promising strategy comprises of drugs coupled with a tumor-targeting bearer that results in wide cytotoxic activity and particular delivery. The B-subunit of Shiga toxin (STxB) is nontoxic and possesses low immunogenicity that exactly binds to the globotriaosylceramide (Gb3/CD77). Gb3/CD77 extremely expresses on a number of human tumors such as pancreatic, colon, and breast cancer and acts as a functional receptor for Shiga toxin (STx). Then, this toxin can be applied to target Gb3-positive human tumors. In this study, we evaluated DT390-STXB chimeric protein as a new anti-tumor candidate via genetically fusing the DT390 fragment of DT538 (Native diphtheria toxin) to STxB.
Methods:
This study intended to investigate the DT390- STxB fusion protein structure in silico. Considering the Escherichia coli codon usage, the genomic construct was designed. The properties and the structure of the protein were determined by an in silico technique. The mRNA structure and the physicochemical characteristics, construction, and the stability of the designed chimeric protein were analyzed using computational and bioinformatics tools and servers. Hence, the GOR4 and I-TASSER online web servers were used to predict the secondary and tertiary structures of the designed protein.
Result:
The results demonstrated that codon adaptation index (CAI) of dt390-stxB chimeric gene raised from 0.6 in the wild type to 0.9 in the chimeric optimized gene. The mfold data revealed that the dt390-stxB mRNA was completely stable to be translated effectively in the novel host. The normal activity of the fusion protein determined by considering the secondary and tertiary structure of each construct. Energy calculation data indicated that the thermodynamic ensemble for mRNA structure was -427.40 kJ/mol. The stability index (SI) of DT390-STxB was 36.95, which is quite appropriate to preserve the stability of the construct. Ultimately, the DT390-STxB was classified as a steady fusion protein according to the Ramachandran plot.
Conclusion:
Our results showed that DT390-STXB was a stable chimeric protein and it can be recruited as a candidate of novel anti-tumor agents for the development of breast cancer treatment.- انتشار مقاله: 16-10-1397
- نویسندگان: Zeynab Mohseni Moghadam,Raheleh Halabian,Hamid Sedighian,Elham Behzadi,Jafar Amani,Abbas Ali Imani Fooladi
- مشاهده
- جایگاه : پژوهشی
- مجله: Iranian Journal of Pathology
- نوع مقاله: Journal Article
- کلمات کلیدی: PCR,ELISA,Brucella melitensis,Brucella abortus
- چکیده:
- چکیده انگلیسی: Background: Rapid diagnosis and differentiation of Brucella is of high importance due to the side effects of antibiotics for the treatment of brucellosis. This study aimed to identify and compare PCR-ELISA as a more accurate diagnositc test with other common molecular and serological tests. Methods: In this experimental and sectional study, during March 2014 to Sep 2015, 52 blood samples of suspected patients with clinical symptoms of brucellosis were evaluated in medical centers all over Iran with serum titers higher than 1:80. Using two pairs of specific primers of Brucella abortus, B. melitensis and DIG-dUTP, Fragment IS711 (The common gene fragment in B. melitensis and B. abortus) was amplified. DIG-ELISA was performed using specific probes of these 2 species of Brucella and patterns were subsequently analyzed, then positive responses were compared by detecting gel electrophoresis. Results: PCR-ELISA method detected all 28 samples from 52 positive samples. Its sensitivity was 6.0 pg concentration of genomic DNA of Brucella. In gel electrophoresis method, 22 samples of all positive samples were detected. PCR-ELISA was more efficient than PCR and bacterial culture method at P-value Conclusion: PCR-ELISA molecular method is more sensitive than other molecular methods, lack of mutagenic color and also a semi-quantitative ability. This method is more effective and more accurate compared to PCR, serology and culture of bacteria. PCR-ELISA does not have false responses. The limitation of this method is detection of bacteria in the genus compared to Multiplex PCR and Gel Electrophoresis.
- انتشار مقاله: 08-05-1394
- نویسندگان: Sharareh Mohammad Hasani,Reza Mirnejad,Vahhab Piranfar,Jafar Amani,Mohamad Javad Vafadar
- مشاهده
- جایگاه : پژوهشی
- مجله: Iranian Journal of Immunology
- نوع مقاله: Journal Article
- کلمات کلیدی: Recombinant vaccine,EHEC,ETEC
- چکیده:
- چکیده انگلیسی: Background: Caused by bacterial, viral, and parasitic pathogens, diarrhea is the second leading cause of death among children under five. Two strains of E. coli, namely Enterotoxigenic, ETEC and Enterohemorrhagic EHEC are the most important causes of this disease in developing countries. EHEC is a major causative agent of bloody diarrhea and hemorrhagic uremic syndrome, while ETEC is the most important cause of diarrhea in neonates and travelers. Objective: To evaluate the immunologic properties of a subunit vaccine candidate comprising the main immunogenic epitopes from these two bacterial strains. Methods: The construct comprised of LTB and CfaB antigens from ETEC, and Intimin and Stx2B antigens from EHEC, was designed, analyzed and synthesized using bioinformatics methods. The chimeric gene was sub-cloned in the expression vector and expressed in E. coli host. The purified chimera protein was injected subcutaneously into the experimental animals. The production of specific antibodies was confirmed by immunological methods, and the protection capacity was evaluated by the challenge of immunized mice with the pathogenic bacteria. Results: Chimeric recombinant protein was able to increase IgG titer. Neutralization assay indicated that the antibodies generated against LtB moiety were able to neutralize ETEC toxin. In animal challenge study, all non-immune mice died within 3 days after the injection of toxin, but all immunized mice survived from Stx toxin. Conclusion: The immunity to both ETEC and EHEC bacteria is significant, and this structure can be considered as a candidate for vaccine production against these bacterial strains.
- انتشار مقاله: 21-08-1397
- نویسندگان: Ahmad Karimi Rahjerdi,Mahyat Jafari,Mohammad Javad Motamedi,Jafar Amani,Ali Hatef Salmanian
- مشاهده
- جایگاه : پژوهشی
- مجله: Iranian Journal of Immunology
- نوع مقاله: Journal Article
- کلمات کلیدی:
- چکیده:
- چکیده انگلیسی: Background: Cholera disease caused by Vibrio cholerae remains a major cause of morbidity and mortality throughout the world. Various strategies with different proteins as immunogens have been tried for vaccine development, none of which have been sufficiently effective to preclude cholera. Chimeric proteins, with their ability to present multiple antigens at the same time, can play important roles in immunization. Objective: To evaluate the immunogenicity of a chimeric construct, comprised of OmpW and CtxB as immunogenic proteins of Vibrio cholera, in BALB/c mice. Methods: The construct was designed after bioinformatics assessments and then expressed in E.coli. Chimeric protein, OmpW, and CtxB were purified with Ni-NTA chromatography and confirmed by Western blotting. Mice were immunized with purified recombinant proteins. The antibody titers and specificity of the immune sera were then analyzed by ELISA and challenged on the pups of immunized mice with 1, 5 and 10 LD50. Mice ileal loop assay was also performed. Results: Significant differences were observed in antibody titers in immunized mice compared to the control groups. Infant mouse challenge was performed so as to compare the protective efficacies of the selected immunogen regimens. Of the Pups from dams immunized with chimeric protein which received 1 LD50, 75% survived. Pups belonging to PBS-immunized dams, experienced 100% mortality. The serum raised toward immunogenic construct, inhibited cholera toxin activity in ileal loop test up to 68%. Conclusion: Chimeric construct is able to induce the immune system and provide up to 75% inhibition of toxin activity against 1 LD50 of Vibrio cholerae.
- انتشار مقاله: 02-07-1397
- نویسندگان: Atina Vakili,Seyed Latif Mousavi Gargari,Shahram Nazarian,Jafar Amani
- مشاهده