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

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

Clinical Chemistry ◽

10.1373/clinchem.2012.200543 ◽

2013 ◽

Vol 59 (7) ◽

pp. 1083-1090 ◽

Cited By ~ 53

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.

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

Molecular & Cellular Proteomics ◽

10.1074/mcp.ra120.002119 ◽

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.

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

Infection and Immunity ◽

10.1128/iai.72.2.863-870.2004 ◽

2004 ◽

Vol 72 (2) ◽

pp. 863-870 ◽

Cited By ~ 21

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.

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

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.6.4.558-566.1999 ◽

1999 ◽

Vol 6 (4) ◽

pp. 558-566 ◽

Cited By ~ 23

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.

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Cyclin B1, D1 overexpression and its correlation with clinical prognostic factors in ovarian carcinoma

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.15067 ◽

2006 ◽

Vol 24 (18_suppl) ◽

pp. 15067-15067

Author(s):

J. W. Kim ◽

Y. T. Kim ◽

H. Y. Kim ◽

M. H. Kang ◽

J. H. Kim ◽

...

Keyword(s):

Cell Cycle ◽

Prognostic Factors ◽

Western Blot ◽

Ovarian Carcinoma ◽

Blot Analysis ◽

Western Blot Analysis ◽

Cyclin B1 ◽

Rt Pcr ◽

The Mean ◽

The Relationship

15067 Background: The change of cell cycle is one of the characteristics of cancer. The various proteins related to the cell cycle have been revealed and their expression in ovarian carcinoma has been demonstrated. Therefore, this study was conducted to determine the expression of cyclin B1, D1 and evaluate the relationship between cyclin B1, D1 and clinical prognostic factors in patients with ovarian carcinoma. Methods: 41 fresh ovarian tissue samples including 36 ovarian carcinomas and 5 normal ovarian tissues were surgically obtained at YUMC from May 2002 to February 2005. Cyclin B1, D1 expression were detected using the quantitative real-time RT-PCR and Western blot analysis. For clinical prognostic factors, age, stage, grade, histopathology, LN metastasis, residual tumor size, CA 125 and DNA flow cytometry were evaluated. Results: By quantitative real time RT-PCR, the mean 2−ΔΔCT value of cyclin B1 and D1 mRNA in ovarian carcinoma was 5.83 ± 12.03, 17.60 ± 22.20, slightly higher than that of the control. (p = 0.67, 0.07). The mean value of relative protein levels of cyclin B1 and D1 in Western blot analysis was also higher in ovarian carcinoma (1.30 ± 0.73, 1.81 ± 1.28, respectively) (p = 0.76, 0.06). No significant relationship of cyclin B1, D1 expression and clinical prognostic factors was observed. Conclusions: The expression of cyclin B1, D1 in ovarian carcinoma was higher than that of the control, although there was no statistical significance. This suggests that cyclin B1, D1 might be involved in the tumorigenesis and the progression of malignancy. Even though there was no significant correlation between cyclin expression and prognostic factors, further studies are needed assessing the relationship between cyclin expression and survival rate to elucidate the role of cyclin as a prognostic factor in ovarian carcinoma. No significant financial relationships to disclose.

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

Clinical and Vaccine Immunology ◽

10.1128/cvi.00388-10 ◽

2010 ◽

Vol 18 (2) ◽

pp. 337-342 ◽

Cited By ~ 36

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.

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

Journal of Helminthology ◽

10.1017/s0022149x15000747 ◽

2015 ◽

Vol 90 (5) ◽

pp. 630-633 ◽

Cited By ~ 2

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.

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

Bangladesh Journal of Medical Microbiology ◽

10.3329/bjmm.v1i1.20491 ◽

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

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

Infection and Immunity ◽

10.1128/iai.66.4.1467-1472.1998 ◽

1998 ◽

Vol 66 (4) ◽

pp. 1467-1472 ◽

Cited By ~ 25

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.

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Antibody validation for Western blot: By the user, for the user

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.010472 ◽

2019 ◽

Vol 295 (4) ◽

pp. 926-939 ◽

Cited By ~ 7

Author(s):

Lakshmi Pillai-Kastoori ◽

Sam Heaton ◽

Steve D. Shiflett ◽

Annabelle C. Roberts ◽

Alejandra Solache ◽

...

Keyword(s):

Western Blot ◽

Western Blotting ◽

Blot Analysis ◽

Western Blot Analysis ◽

Life Sciences ◽

Primary Antibody ◽

Antibody Specificity ◽

Orthogonal Method ◽

Research Findings ◽

Antibody Validation

Well-characterized antibody reagents play a key role in the reproducibility of research findings, and inconsistent antibody performance leads to variability in Western blotting and other immunoassays. The current lack of clear, accepted standards for antibody validation and reporting of experimental details contributes to this problem. Because the performance of primary antibodies is strongly influenced by assay context, recommendations for validation and usage are unique to each type of immunoassay. Practical strategies are proposed for the validation of primary antibody specificity, selectivity, and reproducibility using Western blot analysis. The antibody should produce reproducible results within and between Western blotting experiments and the observed effect confirmed with a complementary or orthogonal method. Routine implementation of standardized antibody validation and reporting in immunoassays such as Western blotting may promote improved reproducibility across the global life sciences community.

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