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Volume 64, Issue 5 (8-2006)
Background: Human plasminogen is a plasma glycoprotein synthesized mainly in the liver. Conversion of plasminogen to plasmin by plasminogen activators is a key event in the fibrinolytic system. In this study, we investigated the effects of two anti-human plasminogen monoclonal antibodies, A1D12 and MC2B8 on Glu-plasminogen activation in presence of u-PA, t-PA and streptokinase.
Methods: Producing of Hybridoma antibodies was performed by fusion of spleen cells from BALB/C mice immunized with Glu-plasminogen and NS1 myeloma cells. Antibody binding to Human Glu-plasminogen was assessed using an ELISA assay. Activation of plasminogen was determined by measuring plasmin generation using the chromogenic substrate S-2251 and the effect of monoclonal antibodies, A1D12 and MC2B8 on plasminogen activation in solution was then evaluated. Initial rates and kinetic parameters of plasminogen activation in the presence of monoclonal antibodies were calculated. The effect of the monoclonal antibody MC2B8 on the rate of plasmin hydrolysis was measured. The effect of F(ab&apos)2 fragment of A1D12 on u-PA catalyzed-plasminogen activation also compared with the effect of the whole antibody in this reaction.
Results: ELISA assay showed that the antibodies reacted well with antigens. A1D12 increased the maximum velocity (Vmax) of plasminogen activation by each of the three plasminogen activators and MC2B8 decreased it. In all activation reactions, the KM value of plasminogen activation did not significantly change in the presence of antibody A1D12 whereas antibody MC2B8 increased the KM value of plasminogen activation by u-PA, fibrin monomer dependent t-PA and streptokinase. Monoclonal antibody MC2B8 had no significant effect on plasmin hydrolysis rate of synthetic substrate S-2251. Activation rate of plasminogen by u-PA in the lower concentration of F (ab)2 fragment of A1D12 was identical to activation in the presence of the whole antibody.
Conclusion: The binding of the A1D12 F(ab) region to Glu-plasminogen increases the catalytic efficiency of plasminogen activation by plasminogen activators. Therefore, it may be useful to apply clinically A1D12 for the therapy of thromboembolic events such as myocardial infarction by humanizing the F(ab) fragment of the A1D12 antibody. Inhibition pattern of antibody MC2B8 obey the mixed type of enzyme inhibition by binding the antibody probably at, or near, the cleavage site of Glu-plasminogen.
Volume 64, Issue 8 (8-2006)
Background: Celiac is one the most common causes of malabsorption and is an autoimmune disorder resulting in damage to intestinal epithelial cells by self T lymphocytes. The main culprit is gluten. The aim of our study was to assess the histopathologic findings of patients suspicious to have celiac disease in their first admission and compare them with serologic findings (endomyosial antibody test: EMA).
Methods: The study is a prospective descriptional type and 95 patients suspicious to have celiac disease were included who went under upper endoscopy and sampling and also serologic studies. Histopathologic findings were grouped by Marsh classification. Specimens in Marsh stage 0 and 1 were also stained for leukocyte common antigen by immunohistochemistry
Results: Ninety five patients, 49 males and 46 females, ranging from 9 months to 17 years (mean: 6.3 ys) were included in the study The most common complaint was abdominal pain. EMA test was positive in 43 patients. Most specimens were categorized as Marsh stage IIIA (51 cases) by histopatholgic examination, which can be seen in many other conditions. Only 16 patients were categorized as Marsh stage IIIB & IIIC, which are seen in established celiac patients No significant correlation was found between histopathologic and serologic findings. No difference was found between H&E staining and immunohistochemical staining in counting of intra- epithelial lymphocytes.
Conclusion: Most of our patients had mild partial villous atrophy which is also seen in many other disorders and also EMA test was negative in this group. It appears that current assessment of celiac disease has many drawbacks and necessity of more specific methods is highly appreciated.
Volume 65, Issue 2 (3-2008)
Background: Until a few decades ago, celiac disease was considered to be essentially a disease of European people and to be very rare in Middle Eastern countries. During the last two decades, having met the criteria for the WHO general screening, the advent and application of novel serological assays used to screen for celiac disease and the use of endoscopic small bowel biopsy have led to increasing numbers of diagnoses of celiac disease in western countries. With this new data, our knowledge on both the clinical pattern and epidemiology of celiac disease has increased, and is now known to be a relatively common autoimmune disorder. Studies performed in different parts of the developing world have shown that the prevalence of celiac disease in this area is similar to or even higher than that in western countries. In fact, celiac disease is known to be the most common form of chronic diarrhea in Iran. However, contrary to common belief, celiac disease is more than a pure digestive alteration. It is a protean systemic disease, and, with a 95 percent genetic predisposition, has a myriad of symptoms including gastrointestinal, dermatological, dental, neurological and behavioral that can occur at a variety of ages. Monosymptomatic, oligosymptomatic, atypical (without gastrointestinal symptoms), silent and latent forms of celiac disease have been identified. In this study we review the epidemiology of celiac disease based on the studies performed in Iran and discuss its pathogenesis, the role of antibodies in the diagnosis of celiac disease and the importance of its diagnosis and treatment in Iran.
Volume 65, Issue 10 (1-2008)
Background: Acute respiratory tract infections, both bacterial and viral, cause 4.5 million childhood deaths worldwide, most of which occur in developing countries. Parainfluenza viruses, of the paramyxoviridae family, are among the common causes of acute respiratory infections, giving rise to 30% of respiratory infections in children before school age. The four parainfluenza viruses that cause a spectrum of respiratory illness in humans are designated as human para influenza virus-1 through 4. Spreading from the respiratory tract by aerosolized secretions or direct hand contact with secretions, parainfluenza viruses replicate in the respiratory epithelium without evidence of systemic spread. The destruction of cells in the upper airways can lead to secondary bacterial invasion and resultant bacterial tracheitis. Eustachian tube obstruction can lead to secondary bacterial invasion of the middle ear space and acute otitis media. In otherwise healthy children, the majority of illnesses remain in the upper respiratory tract. As with many viruses, three approaches to the diagnosis of parainfluenza virus are currently used: viral culture, detection of viral antigen or nucleic acid, and serologic analysis. The gold standard remains the isolation of virus in tissue culture.
Methods: This descriptive case-series study was conducted from January 2003 to January 2004, and included 96 children five years of age and younger. To determine the relative frequency of parainfluenza respiratory tract infection, the nasopharyngeal secretions were studied by immunofluorescent antibody (IFA) assay. Seasonal incidence, age distribution and clinical signs and symptoms of this infection were also recorded.
Results: Among our study group, the relative frequency of parainfluenza respiratory infection was 26%, most commonly in children aged 25-36 months and in autumn. Cough (84%) and rhinorrhea (96%) were the most common symptoms, with fever (68%) as the most common sign in our patients. Pharyngotonsilitis was the most common (40%) clinical manifestation in our patients.
Conclusions: According to above data, patient age and the frequency of parainfluenza infection were similar to other studies.
Volume 66, Issue 4 (7-2008)
Background: The induction of catalytic antibodies (abzymes) was first postulated by Pauling in 1948. Various catalytic antibodies have been detected recently in the sera of patients with several autoimmune pathologies such as systemic lupus erythematosus and rheumatoid arthritis. In addition, antibodies with DNase and RNase activity have been discovered in the milk and sera of healthy human mothers, which shows the physiologic role of these antibodies. In this study, we examined the proteolytic activity of antibodies in the sera of pregnant women.
Methods: IgG antibody fractions were isolated from the sera of 30 healthy pregnant women in the first trimester of pregnancy and 10 control samples (men and nonpregnant women) by subsequent steps of chromatographic purification on Protein G sepharose and sephacryl S-300. All patients were in their first pregnancy and aged 25-35 years. The conditions for proteolytic activity, such as type of buffer, pH and temperature, were optimized. The proteolytic activity of these antibodies was demonstrated by in-gel assay with gelatin as the substrate.
Results: Antibody treatments at the optimum temperature showed that some samples from pregnant women contain proteolytic abzymes, as demonstrated by in-gel assays. Western blot results confirmed that the proteolytic activity is an intrinsic property of the antibodies.
Conclusions: During pregnancy and immediately after delivery women very often experience autoimmune processes similar to those in patients with autoimmune disease. Because of their specific immune status, pregnant women can produce various catalytic antibodies with different enzymatic activity. These proteolytic abzymes might be involved in the direct clearance of antigens from blood.
Volume 66, Issue 8 (11-2008)
Background: Chlamydia Trachomatis is the most common cause of trachoma and subsequently give rise to neonatal chlamydial conjunctivitis (NCC), adult ophthalmic inclusion infection, sexually transmitted diseases (STD) and pneumonia. The goal of this study was to access the incidence of chlamydia trachomatis in the normal (ophthalmic infection free) population.
Methods: In a cross sectional study 250 patients referring to Farabi Eye university Hospital Tehran, Iran for non infectious ophthalmic disease in different age categories were selected and accessed for chlamydial IgM and IgG by ELISA method.
Results: 250 patients (50% men and 50% women) with the mean age of 40 (ranging from one to 83 years old) were tested. IgG was detected in 11 (five females and six males) patients (4.4%) All of them had more than 31 years old. IgM was detected in 18 (13 females and 5 males) patients (7.2%). No test revealed simultaneous high IgG and IgM titre in the same patient.
Conclusions: There was a low grade of chlamydial infection in our study population. So it is recommended to use serological methods for screening of ophthalmic infections in centers where no other test methods are available and in case of positive results confirmatory antigen tests to be used.
Volume 67, Issue 1 (4-2009)
Background: Plasminogen has a central role in fibrinolyrtic system can activate through various activators (PAs) to its active form plasmin and perfoem its vital function that is fibrin clot lysis. Furthermore the fibrinolyrtic system plays a major role in angiogenesis. The fibrinolyrtic system activation control cell migration and invasion. In addition to this, plasmin regulates tumor growth. Monoclonal antibodies, as biological tools, play an important role in basic researches.
Methods: In the first step the effects of antibodies on the activation of fibrinolyrtic system with PAs were evaluated with several methods including macroscopic observation, quantitative measurement of DD/E fragments by D-dimer assay and activation of plasminogen by S-2251 synthetic substrate (ELISA method), subsequently we studied the effect of antibodies on angiogenesis process in an in- vitro model.
Results: Results showed that MC2B8 that is an inhibitor of plasminogen activation in presence of plasminogen activators can inhibit angiogenesis process: A1D12 that is against N-terminal domain of Glu-plasminogen, in addition to activation of fibrinolyrtic system in presence of plasminogen activators, can activate in vitro angiogenesis process.
Conclusion: Plasmin formation is a critical step in invasion and migration of endothelial cells to form new vessels. Plasmin directly participates in angiogenesis by direct fibrin and other matrix components degradation, and indirectly by activating matrix degrading metalloproteinase and angiogenic growth factors. According to the in- vitro results, MC2B8 and A1D12 monoclonal antibodies play roles in this process in a dose dependent manner.
Volume 67, Issue 12 (3-2010)
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Methods: In order to stain above antibody, a phagmid
vector carrying the anti-K99 single-chain Fv
(scFv) antibody was isolated, purified and
transformed into TG1 strain of E.
coli. Afterward,
the antibody was expressed in this cell as phage-scFv
antibody. Phage antibodies were
subsequently eluted, purified and stained with Disperse
Red dye 60
and Coomassie Brilliant
Blue. Finally,
the binding activity of coloured phage antibodies towards the purified K99
fimbriae was verified by immunoblotting.
Results: The results showed that anti-K99
phage antibody was stained with both dyes and the coloured phages were able to
recognize the corresponding antigen.
Conclusions: These protein stains that they usually do not
alter the protein structure can be used for staining phage antibodies. The
coloured phage antibodies retain their binding affinity for the antigens, and
therefore can be applied to detection of relevant antigens.
Volume 68, Issue 5 (8-2010)
Background: M. pneumoniae infection in children is usual and diagnosis of its neurologic complications for rapid treatment is very important. To compare the CSF- M. pneumoniae antibody level between febrile children with acute neurologic signs (Menigoencephalitis, Guillan Barre Syndrome (GBS), Transverse myelitis, Ataxia and so on) with unaffected ones.
Methods: A cross sectional/ case control study in pediatric wards of Rasoul-e-Akram & Mofid hospitals (2007-2009) was done. The amount of Specific M. pneumoniae IgG (ELISA) antibody level determined in CSF of 55 cases and in 10 controls. Chi square values (CI 95%, p< 0.05) calculated for all categorical variables. Sensitivity specificity Positive Predictive Value (PPV) Negative Predictive Value (NPV) of CSF antibody level determined by using the Area under the ROC Curve.Results: Cases (n= 55) aged between five month to 13 years with mean age of 3.84±3.43 years. Area Under Curve (AUC) in ROC was 0.876 (%95 CI, 0.78- 0.96 p< 0.0001). Cut off level for antibody was 0.0025 with 73% sensitivity 90% specificity 100% PPV 28.8% NPV. CSF antibody level had significant difference between cases and controls [0.08± 0.26 Versus 0.001± 0.001 p: 0.02] It had poor agreement between cases and controls (Kappa= 0.27). Lowest amount seen in cases with aseptic meningitis highest amount observed in cases with GBS and cases with focal neurologic signs.
Conclusion: The presence of very low amount (0.0025) of M. pneumoniae antibody in CSF of febrile children with acute neurologic signs had 70% sensitivity and 90% specificity 100% PPV but had low (28.8%) NPV. M. pneumoniae would be a rare cause in cases with aseptic meningitis. Finding the M. pneumoniae-DNAs in CSF are not so frequent (2%) but in high suspicious cases adding this test to determining the CSF antibody level might be helpful.Volume 68, Issue 12 (3-2011)
Background: Alopecia areata, a non-cicatricial form of hair loss, is believed to be an immunologic response that targets hair follicles. Genetic background is important in the pathogenesis of this disorder, although some evidence point to the role of melanocytic antigens. There are some reports on the relationship between alopecia areata and celiac disease. The aim of the present study was to identify antigliadin antibodies in patients with alopecia areata.
Methods: Fifty patients, aged 2.5-50 years, with alopecia areata presenting to the dermatology clinic of Razi Educational Hospital in Tehran, Iran, and fifty healthy individuals, aged 5-48 were matched and enrolled in the study. After signing an informed consent form, blood samples (10 ml clotted blood) were obtained from the participants and sent to referral laboratory for the presence of antigliadin IgA and IgG antibodies. Concentrations of antibodies were measured by ELISA through a full automatic ELISA reader.The data were analyzed statistically.
Results: The study included 29(58%) male and 21(42%) female patients with a mean age of 24.6 years. The control group included 29(58%) male and 21(42%) female individuals with a mean age of 24 years. In the case group, 9(18%) patients were positive for antigliadin antibody, while only one (2%) individual was positive for the antibody in the control group (p<0.001). No other differences were of statistical significance.
Conclusions: Regarding the higher prevalence of antigliadin antibodies in patients with alopecia areata, it would be wise to screen the patients for celiac disease.
Volume 69, Issue 2 (5-2011)
Background: An increased risk for invasive infections with encapsulated bacteria such as Streptococcus pneumoniae has been described in patients with chronic kidney disease (CKD) or in those on dialysis. The aim of this study was to evaluate the antibody response to pneumococcal capsular polysaccharide vaccine in CKD patients. Methods: Sixty-six patients with CKD and 40 healthy individuals were vaccinated with pneumococcal polysaccharide vaccine. The serum antibody response (IgG and IgG2) to the Pneumovax antigens was determined by enzyme-linked immunosorbent assay (ELISA) prior to and four weeks after vaccination. Results: Out of 66 vaccinated patients with CKD, 14 were found to be hyporesponsive to the vaccine (Group 1). Patients with normal specific antibody response were regarded as respondents and were assigned to Group 2 (n=52). The mean post-vaccination IgG titer to the pneumococcal antigens in Group 1 was significantly lower than those in Group 2 (P=0.012 for IgG and P=0.02 for IgG2). The increased anti-pneumococcal IgG titer was significantly lower in patients in Group 1 versus Group 2 (P=0.001) or the healthy control group (P=0.005). During the follow-up period of patients, patients in Group 1 developed higher episodes of pneumococcal infections than those in Group 2 (P=0.007). Conclusion: A substantial proportion of patients with chronic kidney disease fail to mount an adequate antibody response to pneumococcal antigens and remain at significant risk for such infections. These patients should be offered other prophylactic measures to protect them against invasive pneumococcal diseases.
Volume 70, Issue 1 (4-2012)
Background: Diabetes mellitus (DM) is a group of metabolic disorders such as DM I, DM II, secondary causes of DM and gestational diabetes mellitus characterized by hyperglycemic phonotype. The etiology of gestational diabetes mellitus is unknown. Recent studies address the chronic activity of immune system against infections (not autoimmunity) as an important cause of gestational diabetes mellitus. This study aimed to compare T-helper cells 1 and 2 cytokines and associated antibodies in patients with gestational diabetes mellitus and normal pregnant women.
Methods: This cross-sectional study was performed on 45 female patients with GDM and 45 healthy pregnant women in Bandar Abbas, Iran, from 2008- 2009. The exclusion criteria were presence of any infectious diseases or autoimmune disorders such as SLE or RA. Present and past medical histories were taken from the participants thorough physical examination. Blood samples (10 mL) were drawn and sent to laboratory for measuring serum IgE, IgG1, IgG2, IgG3, IgG4, interleukin-10 (IL-10), interleukin-12 (IL-12), transforming growth factor-beta (TGF1), and interferon-gamma (IFN) measurements. T-test and Kolmogorov-Smirnov test were used for data analysis.
Results: The mean age of the patients with GDM and healthy pregnant women was 32.5 and 27.9 yrs, respectively. T-helper 1 and 2 associated antibodies and cytokines had no significant differences between the case and control groups.
Conclusion: The changes in T-helper 1 and 2 associated antibodies and cytokines are not associated with gestational diabetes mellitus and could not be considered as a predictor for gestational diabetes mellitus.
Volume 73, Issue 7 (10-2015)
Background: The influenza virus is one of the most important factors for higher morbidity and mortality in the world. Recently, researchers have been focused on influenza conserved antigenic proteins such as hemagglutinin stalk domain (HA2) for vaccine production and serological studies. The HA2 plays a major role in the fusion of the virus with host cells membrane. The immunity system enables to produce antibody against HA2. The aim of this study is polyclonal antibody production against influenza HA2. Methods: This study was done in the Influenza Research Lab, Pasteur Institute of Iran, Tehran for one year from September 2013 to October 2014. In the present study, recombinant HA2 protein was produced in prokaryotic system and purified using Nickel affinity chromatography. The purified HA2 was mixed with Freund’s adjuvant (complete and incomplete) and injected into two New Zealand white rabbits by intramuscularly and subcutaneously routes. Immunization was continued for several months with two weeks interval. Before each immunization, blood was drawn by venous puncture from the rabbit ear. Function of rabbit's sera was evaluated using radial immunodiffusion (RID) in both forms, Single RID (SRID) and Double RID (DRID). Finally, antiserum activity against HA2 was evaluated using western blotting as serological assay. Results: Sedimentary line and zone was observed in RID assays (SRID and DRID) represent interaction between HA2 protein and anti- HA2 antibody. As well as, western blotting results was positive for HA2 protein. Therefore, these results showed that polyclonal antibody produced against HA2 protein can identify HA2 protein antigenic sites. Conclusion: These findings show that humoral immune responses have properly been stimulated in rabbits and these antibodies can identify HA2 protein and may be suitable for other serological methods.
Volume 74, Issue 1 (4-2016)
Background: Infeliximab is a form of chimeric antibody which neutralizes the most important inflammatory cytokine, TNF-a, in inflammatory disorders. The aim of current study was to pilot expression of chimeric infliximab in Chinese Hamster ovary (CHO) cells.
Methods: In this research study, pVITRO2-neo-mcs vector that consist of infliximab light chain and heavy chain was used to transform into the E.coli by CaCl2 method. The plasmid was then purified and transfected to cultured CHO cells by Lipofectamine 2000® (Invitrogen GmbH, Germany). Transfected cells were selected upon G-418 treatment after 2 weeks and the level of expression, based on standard curve, was measured using IgG ELISA kit after 48 hours for each clone. High level expressed clone was then cultured in roller bottles and recombinant chimeric product was purified by protein A affinity chromatography. The purity of the product was analyzed by 10% gel SDS-PAGE from eluted samples. The efficacy of the purification was analyzed by ELISA before and after purification step. This article is a master's student thesis from February 2015 to August 2016 in pharmaceutical technology development center, Tehran University of Medical Sciences, Tehran, Iran.
Results: The purified plasmid was analyzed on 2% agarose gel. After selective pressure of G-418, 10 stable transfect clones were assessed for infliximab secretion by IgG ELISA kit at 450 nm. The maximum and minimum expression which detected by ELISA were 23 ng/ml and 6 ng/ml, respectively. The band width of infliximab fraction during purification procedure was observed at 0.7-0.8 min. The efficiency of the purification by ELISA was 70%. On SDS-PAGE analysis, two bands, 25 and 50 kDa, respect to light and heavy chains of Infliximab, was confirmed the expression of recombinant protein.
Conclusion: In the current study, the construct for infliximab monoclonal antibody production was designed using genetic engineering techniques and the expression was confirmed by conventional molecular biology methods. The high yield production was carried out in semi-industrial scale using roller bottles with a 70 percentage of purification efficiency.
Volume 75, Issue 8 (11-2017)
Methods: In this experimental study we expressed and purified the recombinant HA1 protein in the second half of 2015 at department of influenza and other respiratory viruses, Pasteur Institute of Iran and then prepared the polyclonal rabbit antibody against it. The vector of pET28aHA1 expressing HA1-His tagged protein of H1N1 influenza A/PR/8/34 virus was used for large scale production of HA1 into E. Coli (BL21). By changing expression conditions such as IPTG (Isopropyl β-D-1-thiogalactopyranoside) concentration, time and temperature of incubation, the expression conditions for HA1 were optimized. The total cell protein harvested and purified by nickel affinity chromatography. All above mentioned experiments monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Results: The efficiency of HA1 recombinant protein was high, equal to 400-600 mg/ml of cell lysate. The polyclonal antibody was prepared by immunizing the rabbits using recombinant HA1 with Freund’s adjuvant according to standard protocols. Efficiency of the antiserum evaluated by enzyme linked immunosorbent assay (ELISA). Determination of antibody level in the collected antiserum using serum-based ELISA showed that the specific antibody has risen well through the immunization schedule.
Conclusion: Our data shows that this polyclonal antibody has potential to be produced in rabbit. It will also be used in the future in influenza diagnosis as well as in other immunological applications such as western blot analyses, immunocytochemistry, and immunohistochemistry.
Volume 76, Issue 11 (2-2019)
Volume 79, Issue 4 (7-2021)
Methods: This study was started in the Pasture Institute of Iran in 2018 September and finished in February 2020. An expression construct containing the human LIV-1 gene was prepared and transferred to the E.coli BL21 by chemical (CaCl2) and heat shock method. The expression of the protein was induced by IPTG and then protein was purified by affinity (Ni-NTA) chromatography. After preparing recombinant protein one female camel was immunized, 6 times at two weeks intervals with Freund's adjuvant. After immunization, the isolated polyclonal antibody was evaluated by ELISA, western blotting and flow cytometry in the detection of LIV-1 protein.
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Results: The result showed that LIV-1 protein was well purified and also the camel polyclonal antibody was able to detect LIV-1 protein in ELISA, western blot and also it can detect LIV-1 on the cell surface as shown by flow cytometry test.
Conclusion: In recent years, LIV-1 has been shown to be a good candidate as a marker in breast cancer, so polyclonal antibodies against LIV-1 can be used for early detection of breast cancer by various diagnostic methods. In this study, it has been shown that polyclonal camel antibodies can be used in laboratory methods and can be considered for immunological tests and therapeutic applications. |
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