scholarly journals Effect of Immunization with Recombinant OspA on Serologic Tests for Lyme Borreliosis

2001 ◽  
Vol 8 (1) ◽  
pp. 79-84 ◽  
Author(s):  
Paul T. Fawcett ◽  
Carlos D. Rose ◽  
Sandra M. Budd ◽  
Kathleen M. Gibney
Keyword(s):  
Western Blot ◽  
Lyme Borreliosis ◽  
Vaccine Recipient ◽  
Enzyme Linked Immunosorbent Assay ◽  
Test Results ◽  
Dot Blot ◽  
Western Blot Assay ◽  
Serologic Tests ◽  
Human Volunteers ◽  
Dot Blot Assay

ABSTRACT This study evaluated the effects of vaccination with OspA on the use of serologic tests as aids in the diagnosis of Lyme borreliosis. Sera from control and OspA-immunized mice and from OspA-immunized human volunteers were tested for serologic reactivity to Borrelia burgdorferi. Testing was performed with samples obtained prior to administration of vaccine and at 30 days following administration of an initial and a second dose of OspA vaccine. The assays used to assess serologic reactivity included an in-house-developed enzyme-linked immunosorbent assay (ELISA), an in-house-developed Western blot assay, two commercial Western blot tests, and a commercially available dot blot assay. Data obtained from this study demonstrate that immunization with the OspA vaccine will cause ELISA to yield positive results (as reported previously) for the majority of vaccine recipients. Results obtained from Western blot analysis indicate that vaccination with recombinant OspA induces production of antibodies which bind to several different borrelial proteins. The degree of reactivity detected by Western blotting varied greatly between the three assays used. The in-house assay showed the least reactivity, while one commercial Western blot test actually yielded positive test results for infection with B. burgdorferi. The usefulness of all three Western blot assays for the diagnosis of potential infection in a vaccine recipient is severely limited by the extensive reactivity caused by vaccination alone. Antibodies produced in response to OspA vaccination did not significantly affect the performance of the dot blot test; thus, it could provide a reliable means to test for infection withB. burgdorferi in OspA vaccine recipients.

A Simple, Rapid and Non-Radiolabeled Immune Assay to Detect Anti-AChR Antibodies in Myasthenia Gravis

2018 ◽  
Vol 50 (3) ◽  
pp. 229-235 ◽  
Author(s):  
Suresh Bokoliya ◽  
Shripad Patil ◽  
Madhu Nagappa ◽  
Arun Taly
Keyword(s):  
Myasthenia Gravis ◽  
Case Control Study ◽  
Enzyme Linked Immunosorbent Assay ◽  
Test Results ◽  
Dot Blot ◽  
Dot Blot Assay ◽  
Healthy Control ◽  
Neurological Illnesses ◽  
Control Study ◽  
Immune Assay

AbstractObjectiveTo assess the practicality of dot-blot testing for rapid and sensitive detection of the antiacetylcholine receptor (anti-AChR) antibodies in myasthenia gravis (MG).MethodsIn this case-control study, we tested serum specimens of 85 patients with MG, 85 healthy control individuals, and 85 patients without MG who have other autoimmune and neurological illnesses. All the serum specimens were tested for anti-AChR antibodies using 3 assays: in-house enzyme-linked immunosorbent assay (ELISA), the dot-blot assay, and commercial ELISA.ResultsIn-house ELISA, commercial ELISA, and dot-blot test results were positive for anti-AChR antibodies in 65 (76.5%) patients with MG. The results of all 3 tests were negative for anti-AChR antibodies in healthy controls and patients without MG. We observed perfect concordance (K = 1, P <.001) between all 3 tests. In-house ELISA correlated significantly (r = 0.873, P <.001) with commercial ELISA. In-house ELISA and the dot-blot test demonstrated similar diagnostic performance in detecting anti-AChR antibodies.ConclusionsThe dot-blot assay is a simple, nonradioactive immune assay for rapid detection of anti-AChR antibodies in MG.

Use of monoclonal and polyclonal antibodies to detect prostatic acid phosphatase by immunoblotting.

1986 ◽  
Vol 32 (10) ◽  
pp. 1832-1835 ◽  
Author(s):  
P C Patel ◽  
L Aubin ◽  
J Côte
Keyword(s):  
Monoclonal Antibodies ◽  
Acid Phosphatase ◽  
Western Blot ◽  
Polyclonal Antibodies ◽  
Prostatic Acid Phosphatase ◽  
The Other ◽  
Dot Blot ◽  
Western Blots ◽  
Dot Blot Assay ◽  
Polyclonal Antisera

Abstract We investigated two techniques of immunoblotting--the Western blot and the dot blot--for use in detecting prostatic acid phosphatase (PAP, EC 3.1.3.2). We used polyclonal antisera to human PAP, produced in rabbits by hyperimmunization with purified PAP, and PAP-specific monoclonal antibodies in the immunoenzymatic protocols. We conclude that PAP can be readily detected by Western blots with use of polyclonal antisera, but not with monoclonal antibodies. On the other hand, using a dot blot assay, we could easily detect PAP with both polyclonal and monoclonal antibodies.

scholarly journals NEAT1 aggravates sepsis-induced acute kidney injury by sponging miR-22-3p

Open Medicine ◽  
2020 ◽  
Vol 15 (1) ◽  
pp. 333-342
Author(s):  
Yawei Feng ◽  
Jun Liu ◽  
Ranliang Wu ◽  
Peng Yang ◽  
Zhiqiang Ye ◽  
...  
Keyword(s):  
Acute Kidney Injury ◽  
Western Blot ◽  
Cell Apoptosis ◽  
Noncoding Rna ◽  
Cell Injury ◽  
Kidney Injury ◽  
Inflammatory Factors ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Western Blot Assay

AbstractBackground and aimAcute kidney injury (AKI) is a common complication of sepsis. Long noncoding RNA nuclear-enriched abundant transcript 1 (NEAT1) plays a vital role in various diseases, including AKI. This study aimed to investigate the function and mechanism of NEAT1 in sepsis-induced AKI.Materials and methodsA septic AKI model was established by treating HK-2 cells with lipopolysaccharide (LPS). The levels of NEAT1 and miR-22-3p were measured by quantitative real-time PCR. Cell apoptosis was assessed by flow cytometry. The levels of apoptosis-related protein and autophagy-related factors were examined by the western blot assay. An enzyme-linked immunosorbent assay was used to calculate the contents of inflammatory factors. The interaction between NEAT1 and miR-22-3p was validated by dual-luciferase reporter assay, RNA immunoprecipitation assay, and RNA pull-down assay. The levels of nuclear factor (NF)-κB pathway-related proteins were evaluated by the western blot assay.ResultsNEAT1 was upregulated, while miR-22-3p was downregulated in patients with sepsis and in LPS-stimulated HK-2 cells. LPS treatment triggered cell apoptosis, autophagy, and inflammatory response in HK-2 cells. NEAT1 knockdown attenuated LPS-induced cell injury. NEAT1 modulated LPS-triggered cell injury by targeting miR-22-3p. Furthermore, NEAT1 regulated the NF-κB pathway by modulating miR-22-3p.ConclusionDepletion of NEAT1 alleviated sepsis-induced AKI via regulating the miR-22-3p/NF-κB pathway.

scholarly journals Elevated Platelet Galectin-3 and Rho-Associated Protein Kinase Activity Are Associated with Hemodialysis Arteriovenous Shunt Dysfunction among Subjects with Diabetes Mellitus

2019 ◽  
Vol 2019 ◽  
pp. 1-9
Author(s):  
Po-Wei Chen ◽  
Ling-Wei Hsu ◽  
Hsien-Yuan Chang ◽  
Ting-Chun Huang ◽  
Jia-Rong Yu ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Western Blot ◽  
Kinase Activity ◽  
Arteriovenous Shunt ◽  
Enzyme Linked Immunosorbent Assay ◽  
Western Blot Assay ◽  
Arteriovenous Shunts ◽  
Galectin 3 ◽  
Shunt Dysfunction ◽  
Rock Activity

Introduction. Hyperglycemia is a major factor in influencing the patency rate of arteriovenous shunts, potentially associated with the RhoA/Rho-associated protein kinase (ROCK) pathway. Besides, galectin-3 mediates thrombotic mechanisms in venous thrombosis and peripheral artery disease. We hypothesized that high ROCK activity and galectin-3 levels are associated with arteriovenous shunt dysfunction. Methods. We prospectively enrolled 38 patients diagnosed with arteriovenous shunt dysfunction. 29 patients received a complete follow-up and each provided two blood samples, which were collected at the first visit for occluded status of arteriovenous shunts and 1 month later for patent status. A Western blot assay for a myosin phosphatase target subunit (MYPT) was performed to examine Rho-kinase activity. A Western blot assay for platelet galectin-3 and enzyme-linked immunosorbent assay (ELISA) for circulating galectin-3 were completed. Results. Higher platelet MYPT ratios and galectin-3 levels were identified at occluded arteriovenous shunts (MYPT ratio: 0.5 [0.3–1.4] vs. 0.4 [0.3–0.6], p = 0.01; galectin-3: 1.2 [0.4–1.6] vs. 0.7 [0.1–1.2], p = 0.0004). The plasma galectin-3 binding protein ELISA was also higher at occluded arteriovenous shunts (8.4 [6.0–9.7] μg/mL vs. 7.1 [4.5–9.1] μg/mL, p = 0.009). Biomarker ratios (occluded/patent status) trended high in patients with poorly controlled diabetes (MYPT ratio: 1.7 [1.0–3.0] vs. 1.1 [0.7–1.3], p = 0.06; galectin-3: 1.6 [1.3–3.4] vs. 1.1 [0.8–1.9], p = 0.05). Conclusion. High platelet ROCK activity and galectin-3 levels are associated with increased risk in arteriovenous shunt dysfunction, especially in patients with poorly controlled diabetes.

scholarly journals Generation and diagnostic application of monoclonal antibodies against Seneca Valley virus

2011 ◽  
Vol 24 (1) ◽  
pp. 42-50 ◽  
Author(s):  
Ming Yang ◽  
Rebekah van Bruggen ◽  
Wanhong Xu
Keyword(s):  
Monoclonal Antibodies ◽  
Indirect Elisa ◽  
Foot And Mouth Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot ◽  
Specific Antibody Response ◽  
Dot Blot Assay ◽  
Infected Cells ◽  
Seneca Valley Virus ◽  
Vesicular Disease

Seneca Valley virus (SVV), a member of the Picornaviridae family, was implicated in a suspicious vesicular disease discovered in pigs from Canada in 2007. Because any outbreak of vesicular disease in pigs is assumed to be foot-and-mouth disease (FMD) until confirmed otherwise, a test for diagnosing the presence of SVV would be a very useful tool. To develop the diagnostic tests for SVV infection, 5 monoclonal antibodies (mAbs) were produced from mice immunized with binary ethylenimine (BEI)-inactivated SVV. Using a dot blot assay, the reactivity of the mAbs was confirmed to be specific for SVV, not reacting with any of the other vesicular disease viruses tested. The mAbs demonstrated reactivity with SVV antigen in infected cells by an immunohistochemistry assay. An SVV-specific competitive enzyme-linked immunosorbent assay (cELISA) was developed using BEI-inactivated SVV antigen and a mAb for serodiagnosis. The cELISA results were compared to the indirect isotype (immunoglobulin [Ig]M and IgG) ELISA and the virus neutralization test. All SVV experimentally inoculated pigs exhibited a positive SVV-specific antibody response at 6 days postinoculation, and the sera remained positive until the end of the experiment on day 57 (>40% inhibition) using the cELISA. The cELISA reflected the profile of the indirect ELISA for both IgM and IgG. This panel of SVV-specific mAbs is valuable for the identification of SVV antigen and the serological detection of SVV-specific antibodies.

scholarly journals Comparison of Diagnostic Techniques for Detecting Tomato Infectious Chlorosis Virus

Plant Disease ◽  
1998 ◽  
Vol 82 (1) ◽  
pp. 84-88 ◽  
Author(s):  
R. H. Li ◽  
G. C. Wisler ◽  
H.-Y. Liu ◽  
J. E. Duffus
Keyword(s):  
Western Blot ◽  
Blot Hybridization ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Dot Blot ◽  
Tomato Plants ◽  
Dot Blot Hybridization ◽  
Field Samples ◽  
Infected Tomato ◽  
Tomato Infectious Chlorosis Virus

A polyclonal antiserum prepared against purified virions of tomato infectious chlorosis virus (TICV) was used to evaluate serological tests for its detection, to determine its distribution in infected plants, to study relationships among isolates of this virus, and to detect it in field samples. A cRNA probe representing TICV RNA 1 and RNA 2 was used in dot blot hybridization tests. A reverse transcriptase-polymerase chain reaction (RT-PCR) assay was also developed for detection of TICV isolates. The comparative study of these four techniques indicated that RT-PCR was 100-fold more sensitive than enzyme-linked immunosorbent assay (ELISA), Western blot, and dot blot hybridization assays for TICV detection. TICV was detected in leaf, stem, flower, and root tissues of the infected tomato plants. However, the virus was not uniformly distributed throughout the infected tomato plants, and the highest viral concentration was observed in fully developed young tomato leaves at the onset of yellowing symptoms. The virus was detected by indirect ELISA, Western blot, dot blot hybridization, and RT-PCR assays in laboratory-infected tomato, tomatillo, potato, and Nicotiana clevelandii and in naturally infected tomato, petunia, and Ranunculus sp. plants obtained from commercial sources. These tests indicate that there are apparently no detectable serological or nucleic acid differences among four TICV isolates obtained from Orange and Yolo Counties of California or from North Carolina or Italy.

scholarly journals Evaluation of Diagnostic Applications of Monoclonal Antibodies against Avian Influenza H7 Viruses

2010 ◽  
Vol 17 (9) ◽  
pp. 1398-1406 ◽  
Author(s):  
Ming Yang ◽  
Alfonso Clavijo ◽  
Jill Graham ◽  
John Pasick ◽  
James Neufeld ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Influenza A ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot ◽  
Western Blots ◽  
Positive Antibody ◽  
Dot Blot Assay ◽  
Diagnostic Applications ◽  
Ha Protein ◽  
H7 Subtype

ABSTRACT A panel of monoclonal antibodies (MAbs) was generated from mice immunized with binary ethylenimine (BEI)-inactivated H7N1 (A/TK/ON/18-2/00) virus. Using a dot blot assay, six of seven MAbs reacted with viruses of the H7 subtype, but not with any of the other 15 hemagglutinin (HA) subtypes tested. Four of the seven MAbs reacted with 14 different H7 isolates, indicating that the MAbs binding epitopes are conserved among viruses of the H7 subtype. The binding epitopes of all seven MAbs were conformational and reacted with the HA1 fraction of the HA protein in Western blots under nonreducing conditions. Applications of these MAbs in the development of rapid tests for H7 subtype viruses were evaluated. The MAbs demonstrated reactivity with AI virus H7 antigen in immunofluorescence and immunohistochemistry assays. Monoclonal antibody 3 showed a very strong immunostaining in the formalin-fixed and paraffin-embedded tissue from the H7N3 virus-infected chicken. A double-antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) was developed using two of the MAbs. The DAS ELISA specifically detected all H7 strains tested in this study. A competitive ELISA (cELISA) for the detection of H7-specific antibodies was evaluated using one MAb and BEI-inactivated H7N1 virus as the antigen. All infected birds showed positive antibody responses at 7 days postinfection. The sensitivity of this cELISA was comparable with that of an influenza A nucleoprotein-based cELISA. This panel of MAbs is valuable in the development of various immunoassays.

scholarly journals Limits in Reliability of Glycoprotein G-Based Type-Specific Serologic Assays for Herpes Simplex Virus Types 1 and 2

1999 ◽  
Vol 37 (2) ◽  
pp. 376-379 ◽  
Author(s):  
D. Scott Schmid ◽  
Denise R. Brown ◽  
Rosane Nisenbaum ◽  
Rae Lyn Burke ◽  
D’Anna Alexander ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Western Blot ◽  
Validation Studies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Western Blot Assay ◽  
Cross Sectional ◽  
Glycoprotein G ◽  
Simplex Virus ◽  
Hsv 1

Type-specific serologic assays for herpes simplex virus (HSV) types 1 and 2 based on glycoprotein G-1 (gG-1) (HSV-1) and gG-2 (HSV-2) discriminate between antibodies against HSV-1 and HSV-2. We previously developed a Western blot assay using gG-1 and gG-2 expressed in baculovirus, performed extensive validation studies, and determined that it was both sensitive and specific for type-specific detection of HSV antibody. Here we report that, among a cohort of Thai military recruits, the serostatus of some individuals changed from positive to negative over time (6.6% among those ever positive for HSV-1, and 14.9% among those ever positive for HSV-2). We tested a subset of these specimens in three other gG-based assays: an enzyme-linked immunosorbent assay, an immunoblot strip assay, and a Western blot assay. Positive-to-negative shifts occurred in every assay; the frequency of the shifts ranged from 6.1% to 21.2% of the specimen sets tested. There was only limited agreement among the assays concerning which individuals lost reactivity. This inaccuracy, exhibited by all of the assay protocols, was not predicted by validation studies employing specimens from cross-sectional studies and was most pronounced in HSV-2 testing. This argues for the inclusion of serial blood specimens in serologic assay validation procedures.

Em18 and Em16, new serologic marker epitopes for alveolar echinococcosis in Western blot analysis, are the only two epitopes recognized by commercially available weak positive (cut off) sera for Em2plus-ELISA

1995 ◽  
Vol 69 (4) ◽  
pp. 369-371 ◽  
Author(s):  
A. Ito ◽  
Y. Osawa ◽  
M. Nakao ◽  
T. Horii ◽  
M. Okamoto ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Alveolar Echinococcosis ◽  
Specific Antigen ◽  
Antibody Responses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Western Blot Assay ◽  
Molecular Weights ◽  
Serologic Marker

AbstractThe assay system for antibody responses against Em2, the most specific antigen for serodiagnosis of alveolar echinococcosis (AE), has been established by enzyme-linked immunosorbent assay (ELISA) but not by Western blot assay, since Em2 antigen is not protein but carbohydrate in nature. Recently we reported that previously undescribed protein epitopes, designated Em18 and Em16 due to their molecular weights, were good serologic markers for AE by Western blot analysis. It has been shown that Em18 and Em16 are the only two epitopes recognized by commercially available weak positive (cut off) sera for the Em2plus-ELISA.

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