scholarly journals Identification of novel serological autoantibodies in Takayasu arteritis patients using HuProt arrays

2020 ◽  
pp. mcp.RA120.002119
Author(s):  
Xiao-Ting Wen ◽  
Guang Song ◽  
Chao-Jun Hu ◽  
Jianbo Pan ◽  
Zi-Yan Wu ◽  
...  
Keyword(s):  
Autoimmune Disease ◽  
Western Blot ◽  
Phase Ii ◽  
Takayasu Arteritis ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Healthy Subjects ◽  
Serum Samples ◽  
Tree Model ◽  
Two Phase

To identify novel autoantibodies of Takayasu arteritis (TAK) using HuProt array-based approach. A two-phase approach was adopted. In Phase I, serum samples collected from 40 TAK patients, 15 autoimmune disease patients, and 20 healthy subjects were screened to identify TAK-specific autoantibodies using human protein (HuProt) arrays. In Phase II, the identified candidate autoantibodies were validated with TAK-focused arrays using an additional cohort comprised of 109 TAK patients, 110 autoimmune disease patients, and 96 healthy subjects. Subsequently, the TAK-specific autoantibodies validated in Phase II were further confirmed using Western blot analysis. We identified and validated eight autoantibodies as potential TAK-specific diagnostic biomarkers, including anti-SPATA7, -QDPR, -SLC25A2, -PRH2, -DIXDC1, -IL17RB, -ZFAND4, and -NOLC1 antibodies, with AUC of 0.803, 0.801, 0.780, 0.696, 0.695, 0.678, 0.635 and 0.613, respectively. SPATA7 could distinguish TAK from healthy and disease controls with 73.4% sensitivity at 85.4% specificity, while QDPR showed 71.6% sensitivity at 86.4% specificity. SLC25A22 showed the highest sensitivity of 80.7%, but at lower specificity of 67.0%. In addition, PRH2, IL17RB and NOLC1 showed good specificities of 88.3%, 85.9% and 86.9%, respectively, but at lower sensitivities (<50%). Finally, DIXDC1 and ZFAND4 showed moderate performance as compared with the other autoantibodies. Using a decision tree model, we could reach a specificity of 94.2% with AUC of 0.843, a significantly improved performance as compared to that by each individual biomarker. The performance of three autoantibodies, namely anti-SPATA7, -QDPR and -PRH2, were successfully confirmed with Western blot analysis. Using this two-phase strategy, we identified and validated eight novel autoantibodies as TAK–specific biomarker candidates, three of which could be readily adopted in a clinical setting.

Lav/HTLV-III Neutralizing Antibodies in the Sera of Patients with Aids, Lymphadenopathy Syndrome and Asymptomatic Seropositive Individuals

1986 ◽  
Vol 72 (3) ◽  
pp. 219-224 ◽  
Author(s):  
Georgine P. Faulkner-Valle ◽  
Anita De Rossi ◽  
Oriella Dalla Gassa ◽  
Luigi Chieco-Bianchi
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Neutralizing Antibodies ◽  
Elisa Test ◽  
Antibody Specificity ◽  
Serum Samples ◽  
Neutralizing Activity ◽  
The Mean ◽  
Asymptomatic Individuals

Serum samples which had previously been found positive for LAV/HTLV-III antibodies by the ELISA test and then confirmed by radioimmunoassay (Western blot) were tested for the presence of neutralizing antibodies. No neutralizing activity was found in the sera of a group of patients with the clinical diagnosis of AIDS. However in patients with LAS and other related pathologic conditions the percentage of sera positive for neutralizing antibodies was 27 % and 55 % respectively. At least 50 % of the sera from seropositive asymptomatic individuals had neutralizing activity but with the exception of the haemophiliac group the mean titre was much lower than that of LAS patients. No relationship was found between the neutralizing titre and the antibody specificity detected by Western blot analysis for p41 and p120.

scholarly journals Time-Dependent Degradation Pattern of Cardiac Troponin T Following Myocardial Infarction

2013 ◽  
Vol 59 (7) ◽  
pp. 1083-1090 ◽  
Author(s):  
Eline PM Cardinaels ◽  
Alma MA Mingels ◽  
Tom van Rooij ◽  
Paul O Collinson ◽  
Frits W Prinzen ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Western Blot ◽  
Blot Analysis ◽  
Cardiac Troponin ◽  
Western Blot Analysis ◽  
Troponin T ◽  
Time Dependent ◽  
Cardiac Troponin T ◽  
Serum Samples ◽  
Degradation Pattern

BACKGROUNDCardiac troponin T (cTnT) is widely used for the diagnosis of acute myocardial infarction (AMI). However, it is still unclear whether degraded cTnT forms circulate in the patient's blood. We therefore aimed to elucidate which cTnT forms are detected by the clinical assay.METHODSSeparation of cTnT forms by gel filtration chromatography (GFC) was performed in sera from 13 AMI patients to examine cTnT degradation. The GFC eluates were subjected to Western blot analysis with the original antibodies from the Roche immunoassay used to mimic the clinical cTnT assay. To investigate the degradation pattern with time, standardized serum samples of 18 AMI patients collected 0–72 h after admission were analyzed by Western blot analysis.RESULTSGFC analysis of AMI patients' sera revealed 2 cTnT peaks with retention volumes of 5 and 21 mL. Western blot analysis identified these peaks as cTnT fragments of 29 and 14–18 kDa, respectively. Furthermore, the performance of direct Western blots on standardized serum samples demonstrated a time-dependent degradation pattern of cTnT, with fragments ranging between 14 and 40 kDa. Intact cTnT (40 kDa) was present in only 3 patients within the first 8 h after hospital admission.CONCLUSIONSThese results demonstrate that the Roche cTnT immunoassay detects intact as well as degraded cTnT forms in AMI patients' sera during the period of diagnostic testing. Moreover, following AMI, cTnT is degraded in a time-dependent pattern.

scholarly journals Characterization of Immunoglobulin G and Its Subclass Response to Indian Kala-Azar Infection before and after Chemotherapy

2004 ◽  
Vol 72 (2) ◽  
pp. 863-870 ◽  
Author(s):  
Rajesh Ravindran ◽  
Khairul Anam ◽  
Bibhas C. Bairagi ◽  
Bibhuti Saha ◽  
Netai Pramanik ◽  
...  
Keyword(s):  
Western Blot ◽  
Immunoglobulin G ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Igg Subclasses ◽  
Cross Reaction ◽  
Future Application ◽  
Healthy Controls ◽  
Serum Samples ◽  
Kala Azar

ABSTRACT Serologic parameters of kala-azar were evaluated by Western blot analysis. Sera from kala-azar patients with confirmed diagnoses were screened for immunoglobulin G (IgG) and IgG subclass-specific reactivity against Leishmania donovani membrane antigen (LAg). Heterogenous LAg-specific IgG reactivity with numerous proteins with molecular masses ranging from 18 to 190 kDa was observed. Though the individual band patterns were varied, seven polypeptides of approximately 31, 34, 51, 63, 72, 91, and 120 kDa were immunoreactive with all the sera tested from kala-azar patients. The band patterns of the immunoblots of sera from patients after treatment and clinical cure with sodium antimony gluconate revealed a decrease in the frequency of the bands. Still, recognition of the 63- and 120-kDa bands was 100%, and the 55- and 91-kDa fractions were recognized in 93% of the sera from cured individuals. Among the IgG subclasses, IgG1 reacted with the greatest number of polypeptides. The 63-kDa protein was again detected by all of the IgG subclasses of all the sera tested. Other fractions recognized by the subclasses of more than 70% of the serum samples included those of 47, 51, 55, and 78 kDa. Following treatment, 63- and 51-kDa bands were the most reactive with the IgG subclasses. LAg-associated cross-reaction with other reference human antisera revealed a mild reactivity of the 63-kDa polypeptide with some of the serum samples from leprosy, malaria, typhoid, tuberculosis, and healthy controls. Western blot analysis of LAg entrapped in liposomes, strong vaccine candidates against experimental visceral leishmaniasis, revealed a more restricted band pattern. The 63-kDa fraction revealed by all pre- and posttreatment sera showed almost negligible levels of cross-reaction with sera from patients with other diseases or from healthy controls. These observations provide insight into induced immunity during kala-azar infection for future application.

scholarly journals Identification of Bartonella-Specific Immunodominant Antigens Recognized by the Feline Humoral Immune System

1999 ◽  
Vol 6 (4) ◽  
pp. 558-566 ◽  
Author(s):  
R. L. Freeland ◽  
D. T. Scholl ◽  
K. R. Rohde ◽  
L. J. Shelton ◽  
K. L. O’Reilly
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Bartonella Henselae ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Whole Cells ◽  
Humoral Immune ◽  
Antibody Titers ◽  
Specific Antigens

ABSTRACT The seroreactivities of both naturally and experimentally infected cats to Bartonella henselae was examined. Serum samples collected weekly from nine cats experimentally infected with B. henselae LSU16 were tested by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. The magnitude and isotype of the antibody response were investigated by ELISA. Western blot analysis allowed the identification of at least 24Bartonella-specific antigens recognized by the cats during infection. Antibody titers to specific antigens, as determined by Western blot analysis, ranged from 10 to 640 and varied among the different antibody-antigen interactions. Absorption of sera from an experimentally infected cat, using whole cells and cell lysates of various Bartonella species and other bacteria that commonly colonize cats, supported the identification of thoseBartonella-specific antigens recognized by the experimentally infected cats. Furthermore, a number of possible species- and type-specific antigens were identified. Finally, sera obtained from cats at local animal shelters were screened for the presence of antibodies directed against theBartonella-specific bands identified in the experimentally infected cats. A number of Bartonella-specific antigens have been identified to which strong antibody responses are generated in both experimentally and naturally infected cats, some of which may be useful in diagnosing species- and/or type-specific infections. In addition, the results from these experiments will lead to the development of monoclonal antibodies targeted against those genus-, species-, and type-specific antigens.

scholarly journals Spherical Body Protein 4 Is a New Serological Antigen for Global Detection ofBabesia bovisInfection in Cattle

2010 ◽  
Vol 18 (2) ◽  
pp. 337-342 ◽  
Author(s):  
Mohamad Alaa Terkawi ◽  
Nguyen Xuan Huyen ◽  
Putut Eko Wibowo ◽  
Faasoa Junior Seuseu ◽  
Mahmoud Aboulaila ◽  
...  
Keyword(s):  
Western Blot ◽  
Recombinant Proteins ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Fluorescent Antibody ◽  
Spherical Body ◽  
Babesia Bovis ◽  
Serum Samples ◽  
Body Protein ◽  
Geographical Regions

ABSTRACTFiveBabesia bovisrecombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), C-terminal rhoptry-associated protein 1 (BbRAP-1/CT), truncated thrombospondin-related anonymous protein (BbTRAP-T), spherical body protein 1 (BbSBP-1), and spherical body protein 4 (BbSBP-4), were evaluated as diagnostic antigens to detect the infection in cattle. The recombinant proteins were highly antigenic when tested with experimentallyB. bovis-infected bovine serum in Western blot analysis. Furthermore, five antisera that had been raised against each of the recombinant proteins reacted specifically with the corresponding authentic protein, as determined in Western blot analysis. Next, enzyme-linked immunosorbent assays (ELISAs) using these recombinant proteins were evaluated for diagnostic use, and the sensitivity and specificity of each protein were demonstrated with a series of serum samples from experimentallyB. bovis-infected cattle. Furthermore, a total of 669 field serum samples collected from cattle in regions ofB. bovisendemicity in seven countries were tested with the ELISAs, and the results were compared to those of an indirect fluorescent antibody test (IFAT), as a reference. Among five recombinant antigens, recombinant BbSBP-4 (rBbSBP-4) had the highest concordance rate (85.3%) and kappa value (0.705), indicating its reliability in the detection of specific antibodies toB. bovisin cattle, even in different geographical regions. Overall, we have successfully developed an ELISA based on rBbSBP-4 as a new serological antigen for a practical and sensitive test which will be applicable for epidemiologic survey and control programs in the future.

scholarly journals Performance of Tsol-p27 antigen for the serological diagnosis of cysticercosis in Mozambique

2015 ◽  
Vol 90 (5) ◽  
pp. 630-633 ◽  
Author(s):  
N. Nhancupe ◽  
E.V. Noormahomed ◽  
S. Afonso ◽  
K.I. Falk ◽  
J. Lindh
Keyword(s):  
Western Blot ◽  
Sensitivity And Specificity ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Low Cost ◽  
High Sensitivity ◽  
Diagnostic Methods ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Neuroimaging Techniques

AbstractThe diagnosis of neurocysticercosis (NCC) requires expensive neuroimaging techniques that are seldom affordable for people in endemic countries. Accordingly, there is a need for new low-cost diagnostic methods that offer high sensitivity and specificity. In this study, we evaluated Western blot analysis of the previously described recombinant antigen Tsol-p27 in relation to a commercial or in-house enzyme-linked immunosorbent assay (ELISA) for NCC, and compared the results with those provided by a commercial enzyme-linked immunoelectrotransfer blot (EITB) assay, which was regarded as the reference standard method. The analysed serum samples were obtained from 165 people, 18 of whom were confirmed to be NCC positive by EITB. Comparing our Western blot analysis of Tsol-p27 with a previous evaluation performed in Central America showed similar specificity (96.69% versus 97.8%) and sensitivity (85.71% versus 86.7%). The present results indicate that the recombinant Tsol-p27 antigen provides good sensitivity and specificity, and might be preferable as a diagnostic antigen in poorly equipped laboratories in endemic countries.

scholarly journals Comparison of Western Blot Technique with Microscopy and Culture for Diagnosis of Tuberculosis

2016 ◽  
Vol 1 (1) ◽  
pp. 21-24
Author(s):  
Md. Jamal Uddin Gazi ◽  
Md. Ruhul Amin Miah ◽  
Sharmeen Ahmed ◽  
Abu Naser Ibne Sattar
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Antibody Response ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Polyacrylamide Gel ◽  
Serum Samples ◽  
Bacteriological Diagnosis ◽  
Blot Technique ◽  
Study Population

Diagnosis of tuberculosis is usually done by microscopy for AFB and other tests. But each test has different limitation. This study was carried out to compare western blot technique with microscopy and culture for diagnosis of tuberculosis. Sputum and other samples were collected from 112 TB patients for bacteriological diagnosis by microscopy and culture for AFB. Serum samples were collected from 112 TB patients and 50 control subjects for detection of antibody response by western blot. For western blot analysis, Mycobacterium tuberculosis sonicate antigen extract was fractionated by electrophoresis on  polyacrylamide gel.  Western blot analysis revealed four polypeptides against which antibody response of study population were observed. The sensitivity of western blot was 73.21% which was higher than that of microscopy (63.39%) and culture (57.14%) for diagnosis of tuberculosis. The specificity of western blot was 92%.Bangladesh J Med Microbiol 2007; 01 (01):21-24

scholarly journals Molecular Analysis of Shiga ToxigenicEscherichia coli O111:H− Proteins Which React with Sera from Patients with Hemolytic-Uremic Syndrome

1998 ◽  
Vol 66 (4) ◽  
pp. 1467-1472 ◽  
Author(s):  
Elena Voss ◽  
Adrienne W. Paton ◽  
Paul A. Manning ◽  
James C. Paton
Keyword(s):  
Western Blot ◽  
Hemolytic Uremic Syndrome ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Proteinase K ◽  
Serum Samples ◽  
Healthy Human ◽  
Fermented Sausage ◽  
Convalescent Phase ◽  
Uremic Syndrome

ABSTRACT Western blot analysis was used to assess the reactivity of convalescent-phase sera from patients who were associated with an outbreak of hemolytic-uremic syndrome (HUS) caused by fermented sausage contaminated with Shiga toxin-producing Escherichia coli(STEC). The predominant STEC isolated from HUS patients belonged to serotype O111:H−, and reactivity to O111:H−whole-cell lysates, treated or untreated with proteinase K, was examined. As expected, all five serum samples demonstrated a marked anti-lipopolysaccharide response, but several protein bands were also immunoreactive, particularly one with an apparent size of 94 kDa. One convalescent-phase serum sample was subsequently used to screen an O111:H− cosmid bank and 2 of 900 cosmid clones were found to be positive, both of which contained a similar DNA insert. Western blot analysis of one of these clones identified three major immunoreactive protein bands of approximately 94, 70, and 50 kDa. An immune response to the three proteins was detectable with all five convalescent-phase serum samples but not with healthy human serum. Immunoreactive 94- and 50-kDa species were produced by a deletion derivative of the cosmid containing a 7-kb STEC DNA insert. Sequence analysis of this region indicated that it is part of the locus for enterocyte effacement, including the eaeA gene which encodes intimin. The deduced amino acid sequence of the O111:H− intimin was 88.6% identical to intimin from O157:H7 STEC, and the most divergent region was the 200 residues at the carboxyl terminus, which were only 75% identical. Such variation may be antigenically significant as serum from a HUS patient infected only with the O111:H− STEC reacted with intimin from an enteropathogenic E. coli O111 strain, as well as several other eaeA-positive STEC isolates, but not with aneaeA-positive STEC belonging to serotype O157:H−. Sera from two of the other HUS patients also failed to react with intimin from this latter strain. However, intimin from O157:H− STEC did react with serum from a patient infected with both O111:H− and O157:H− STEC.

Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

2010 ◽  
Vol 113 (Special_Supplement) ◽  
pp. 228-235 ◽  
Author(s):  
Qiang Jia ◽  
Yanhe Li ◽  
Desheng Xu ◽  
Zhenjiang Li ◽  
Zhiyuan Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Gamma Knife ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

Synthesis and Characterization of Quinoline-3-Carboxamide Derivatives as Inhibitors of the ATM Kinase

2020 ◽  
Vol 20 (23) ◽  
pp. 2070-2079
Author(s):  
Srimadhavi Ravi ◽  
Sugata Barui ◽  
Sivapriya Kirubakaran ◽  
Parul Duhan ◽  
Kaushik Bhowmik
Keyword(s):  
Western Blot ◽  
Cancer Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cancer Cell Lines ◽  
Atm Kinase ◽  
Cytotoxicity Studies

Background: The importance of inhibiting the kinases of the DDR pathway for radiosensitizing cancer cells is well established. Cancer cells exploit these kinases for their survival, which leads to the development of resistance towards DNA damaging therapeutics. Objective: In this article, the focus is on targeting the key mediator of the DDR pathway, the ATM kinase. A new set of quinoline-3-carboxamides, as potential inhibitors of ATM, is reported. Methods: Quinoline-3-carboxamide derivatives were synthesized and cytotoxicity assay was performed to analyze the effect of molecules on different cancer cell lines like HCT116, MDA-MB-468, and MDA-MB-231. Results: Three of the synthesized compounds showed promising cytotoxicity towards a selected set of cancer cell lines. Western Blot analysis was also performed by pre-treating the cells with quercetin, a known ATM upregulator, by causing DNA double-strand breaks. SAR studies suggested the importance of the electron-donating nature of the R group for the molecule to be toxic. Finally, Western-Blot analysis confirmed the down-regulation of ATM in the cells. Additionally, the PTEN negative cell line, MDA-MB-468, was more sensitive towards the compounds in comparison with the PTEN positive cell line, MDA-MB-231. Cytotoxicity studies against 293T cells showed that the compounds were at least three times less toxic when compared with HCT116. Conclusion: In conclusion, these experiments will lay the groundwork for the evolution of potent and selective ATM inhibitors for the radio- and chemo-sensitization of cancer cells.

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