Comparison Of Four Anti-Avian IgY Secondary Antibodies Used In Western Blot And Dot-Blot ELISA To Detect Avian Bornavirus Antibodies In Four Different Bird Species

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

Clinical Chemistry ◽

10.1093/clinchem/32.10.1832 ◽

1986 ◽

Vol 32 (10) ◽

pp. 1832-1835 ◽

Cited By ~ 2

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.

Lenalidomide Induces Lipid Raft Assembly to Enhance Erythropoietin Receptor Signaling in Myelodysplastic Syndrome Progenitors

Blood ◽

10.1182/blood.v124.21.4623.4623 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4623-4623

Author(s):

Kathy L McGraw ◽

Ashley A Basiorka ◽

Joseph Johnson ◽

Justine Clark ◽

Gisela Caceres ◽

...

Keyword(s):

Bone Marrow ◽

Confocal Microscopy ◽

Western Blot ◽

Lipid Rafts ◽

Lipid Raft ◽

Erythropoietin Receptor ◽

Dot Blot ◽

Erythroid Progenitors ◽

Lyn Kinase ◽

Erythroid Precursors

Abstract Erythropoietin receptor (EpoR) signaling is impaired in patients with Myelodysplastic Syndromes (MDS) despite appropriate growth factor production and cellular receptor display. We previously reported that EpoR signaling is dependent upon receptor localization within membrane lipid raft microdomains, and that disruption of raft integrity abolishes signaling capacity (McGraw KL, et al. PLoS One 2012). Here, we show that MDS erythroid progenitors display markedly diminished raft assembly (p=0.005) and smaller raft aggregates (p=0.023) compared to normal controls. Because lenalidomide triggers raft coalescence in T-lymphocytes to promote immune synapse formation, we assessed the effects of lenalidomide on raft assembly in MDS erythroid precursors and UT7 cells. Lipid rafts were isolated from UT7 cells using ultracentrifugation and identified by GM-1 dot blot and Lyn kinase western blot. Lenalidomide rapidly induced lipid raft formation in UT7 cells which was confirmed by confocal microscopy visualization of GM-1 fluorescence. Lenalidomide also significantly induced lipid raft formation in pooled MDS erythroid progenitors (CD71+, cKit+) from 11 patients [mean raft size, control (n=569) vs. lenalidomide treatment (n=659), p<0.001], with no significant change observed in pooled erythroids from 3 normal donors (n= 327 for control and n=365 for lenalidomide treated, p=0.37). Interestingly, lipid rafts were significantly larger in erythroid progenitors from patients who responded (n=5) to lenalidomide treatment compared to non-responders (n=3) (75.52 ±13.68 vs. 35.85 ±8.56, p=0.02). Although lenalidomide increased raft size in erythroid progenitors from both responders (p=0.0007) and non-responders (p=0.013), mean raft size was greater in erythroid precursors from responding patients after treatment (p=0.11). Increased raft aggregation after lenalidomide treatment was accompanied by EpoR recruitment into raft fractions together with STAT5, JAK2, and Lyn kinase, whereas the JAK2 phosphatase, CD45, a negative regulator of EpoR signaling, was displaced from raft fractions. Incubation with lenalidomide prior to Epo stimulation enhanced both JAK2 and STAT5 phosphorylation in UT7 cells and primary MDS erythroid precursors. Bone marrow specimens from 12 non-del(5q) IPSS lower risk, lenalidomide naive MDS patients were analyzed by flow cytometry to compare changes in STAT5 phosphorylation in response to Epo stimulation in the presence or absence of lenalidomide. We found a 79.1% mean increase in p-STAT5 mean fluorescence intensity (MFI 95th percentile) in CD45dim, CD71high, Glylow erythroid precursors in 7 of the 12 patient specimens following lenalidomide exposure. Furthermore, increased STAT5 phosphorylation was accompanied by increased DNA binding of the transcription factor in UT7 cells, and improved erythroid colony forming capacity in both UT7 and primary MDS bone marrow cells. Raft induction was associated with F-actin polymerization that was blocked by Rho kinase inhibition and confirmed by lipid raft isolation followed by dot blot with western blot and confocal microscopy. These data provide new insight into abnormalities in the EpoR signaling platform that underlie impaired Epo responsiveness in MDS erythroid precursors. Our findings that deficient raft integrity impairs EpoR signaling provides a novel strategy to enhance EpoR signal fidelity in non-del(5q) MDS. These data also warrant investigation in a larger data set to determine whether lipid raft size may be a predictive biomarker for lenalidomide response. Disclosures List: Celgene: Consultancy.

82 Western blot and dot blot analyses of immunologic cross-reactivity in legume allergic patients

Journal of Allergy and Clinical Immunology ◽

10.1016/0091-6749(88)90318-1 ◽

1988 ◽

Vol 81 (1) ◽

pp. 189

Author(s):

JB Broadbent ◽

S Taylor ◽

EH Adam ◽

HA Sampson

Keyword(s):

Western Blot ◽

Cross Reactivity ◽

Dot Blot

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

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.8.1.79-84.2001 ◽

2001 ◽

Vol 8 (1) ◽

pp. 79-84 ◽

Cited By ~ 3

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.

Comparison of Diagnostic Techniques for Detecting Tomato Infectious Chlorosis Virus

Plant Disease ◽

10.1094/pdis.1998.82.1.84 ◽

1998 ◽

Vol 82 (1) ◽

pp. 84-88 ◽

Cited By ~ 24

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.

Immunoblotting Detection of Immunoglobulin G Post Subcutaneous Immunization Of Protein Hemaglutinin Pili Klebsiella pneumoniae 12,8 kDa on Mice BALB / C

Journal of Agromedicine and Medical Sciences ◽

10.19184/ams.v3i2.5069 ◽

2017 ◽

Vol 3 (2) ◽

pp. 40 ◽

Cited By ~ 1

Author(s):

Dini Agustina

Keyword(s):

Klebsiella Pneumoniae ◽

Western Blot ◽

Immunoglobulin G ◽

Quantitative Result ◽

Protein Subunit ◽

Dot Blot ◽

Humoral Immune ◽

Hemagglutinin Protein ◽

Encapsulated Bacteria ◽

Subcutaneous Immunization

Klebsiella pneumoniae is the second most common cause of community infection and nosocomial infections due to Gram-negative bacteria. These bacteria are able to induce the onset of immune response, especially humoral immune response.Humoral immunity acts through the activation of B cells that produce antibodies. Antibodies, especially IgG, will cause encapsulated bacteria such as Klebsiella pneumoniae to be better phagocytosed. The purpose of this study was to determine the IgG response to hemagglutinin protein pili K. pneumoniae 12.8 kDa. The method used in this research is Immunoblotting method with western blot and dot blot. The primary antibodies used for the western blot and dot blot tests were isolated from BALB / C mice serum induced with the subcutaneous pili K. pneumoniae 12.8 kDa protein. To get the standard in assessing the results of dot blot were used Corel Photo-paint X6. The semi-quantitative result of dot blot was obtained with the strongest reaction of the antibody dilution at 1/100 while the antigen dilution titer at 1 / 10.000. Results from western blotting showed a positive reaction of the pili protein subunit with a molecular weight of 128.1 kDa, 114.4 kDa, 64.9 kDa, 31.1 kDa, 27.7 kDa, 24.8 kDa, 20.9 kDa, 12.8 kDa, and 10 kDa. The conclusion of this study is the immunization of hemagglutinin pili K. pneumoniae 12.8 kDa subcutaneously capable of inducing the formationof Immunoglobulin G in BALB / C mice.

Hemagglutinating and acid phosphatase (AcPASE) activities in developing seedlings of four species of Cucurbitaceae

Acta Societatis Botanicorum Poloniae ◽

10.5586/asbp.1999.041 ◽

2014 ◽

Vol 68 (4) ◽

pp. 291-297

Author(s):

Irena Lorenc-Kubis ◽

Urszula Uram-Walaszczyk

Keyword(s):

Acid Phosphatase ◽

Western Blot ◽

Cucumis Sativus ◽

Cucurbita Pepo ◽

Cucurbita Maxima ◽

Dot Blot ◽

Con A ◽

Seeds Germination

The acid phosphatase and hemagglutinating activities of four species of Cucurbitaceae were determined during seeds germination and seedlings development. In all cases traces of enzyme and hemagglutinating activities were found in dry and imbibided seeds. In developing seedlings of Cucumis sativus the activities increased to maximum on the 3rd day while in other species on the 6th day of germination and than fell down. Dot blot and Western blot techniques have shown that in seeds and seedlings of all investigated species present were proteins which cross-reacted with antibodies raised against lectins: CLBa and Con A. It has been shown that proteins from seeds and seedlings of Cucurbita maxima var. bambino, Cucurbita pepo var. giromontia and Cucumis sativus had more pronounced antigenical similarity to lectin CLBa (from Cucurbitaceae) than Con A, while proteins from cotyledons of Cucurbita pepo var. patissonina reacted better with antibodies raised against Con A (the lectin from Papilionaceae) than with CLBa.

Western Blotting (WB) Technique Guideline for Separation and Isolation of Protein

BioScientia Medicina ◽

10.32539/bsm.v5i2.229 ◽

2021 ◽

Vol 5 (2) ◽

pp. 359-372

Author(s):

Rachmat Hidayat ◽

Patricia Wulandari

Keyword(s):

Western Blot ◽

Western Blotting ◽

Complex Mixture ◽

Solid Support ◽

Target Proteins ◽

Secondary Antibodies

A B S T R A C TWestern blotting is an important technique used in cell and molecularbiology. Using the western blot, researchers can identify specific proteinsfrom the complex mixture of proteins extracted from cells. This techniqueuses three elements to accomplish this task: (1) separation by size, (2)transfer to a solid support, and (3) marking target proteins using appropriateprimary and secondary antibodies to visualize. This paper will attempt toexplain the techniques and theory behind western blot, and offer severalways to solve the problem

Serological follow-up of patients with paracoccidioidomycosis treated with itraconazole using Dot-blot, ELISA and Western-blot

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46651997000500004 ◽

1997 ◽

Vol 39 (5) ◽

pp. 261-270 ◽

Cited By ~ 16

Author(s):

Rosana MARTINS ◽

Sílvio MARQUES ◽

Marino ALVES ◽

Denise FECCHIO ◽

Marcello F. de FRANCO

Keyword(s):

Western Blot ◽

Humoral Response ◽

Dot Blot ◽

Serological Tests ◽

Molecular Weights ◽

Igm Antibodies ◽

Antibody Titers ◽

Pre Treatment ◽

Post Therapy

Twenty-seven mycologically proven cases of paracoccidioidomycosis (PCM) were treated with itraconazole (100-200 mg/day in month 1 and 100 mg/day until month 6-8) and evaluated clinically and serologically, up to 3.5 years post-therapy, using Dot-blot and ELISA for measuring the titers of IgG, IgA and IgM anti-P. brasiliensis antibodies and Western-blot for determining IgG, IgA and IgM antibodies against the antigen components of the fungus. Before treatment, 81.5% (Dot-blot) and 84% (ELISA) of the patients presented elevated IgG anti-P. brasiliensis antibody titers which dropped slightly with treatment. On the other hand, the percentages of pre-treatment high-titered sera for IgA and IgM anti-P.brasiliensis were lower (5l.9% and 5l.8%: Dot-blot; 16.5 and 36%: ELISA, respectively) but the titers tended to become negative more frequently with treatment. Prior to treatment, the percentages of positivity for IgG, IgA and IgM anti-P.brasiliensis antibodies in Western-blot were 96%, 20.8% and 41.6%, respectively. Antigens with molecular weights varying from 16-78 kDa, from 21-76 kDa and from 27-78 kDa were reactive for IgG, IgA and IgM antibodies, respectively. The most frequently reactive antigenic components had molecular weights of 27, 33 and 43 kDa for IgG, and 70 for IgA and IgM antibodies. During the period of study, the patients responded well to treatment. The present data confirm the diversity and complexity of the humoral response in PCM, and the importance of utilizing different serological tests to detect IgG, IgA and IgM anti-P. brasiliensis antibodies

Identification of a Human Lactoferrin-Binding Protein in Gardnerella vaginalis

Infection and Immunity ◽

10.1128/iai.68.6.3443-3447.2000 ◽

2000 ◽

Vol 68 (6) ◽

pp. 3443-3447 ◽

Cited By ~ 10

Author(s):

Gregory P. Jarosik ◽

Carol Beth Land

Keyword(s):

Western Blot ◽

Blot Analysis ◽

Binding Protein ◽

Sole Source ◽

Binding Activity ◽

Human Lactoferrin ◽

Gardnerella Vaginalis ◽

Dot Blot ◽

Heat Stable ◽

Dot Blot Assay

ABSTRACT Previous studies have shown that Gardnerella vaginaliscan utilize iron-loaded human lactoferrin as a sole source of iron. In this study, G. vaginalis cells were shown to bind digoxigenin (DIG)-labeled human lactoferrin in a dot blot assay. Using the DIG-labeled human lactoferrin, a 120-kDa human lactoferrin-binding protein was detected by Western blot analysis of G. vaginalis proteins. The lactoferrin-binding activity of this protein was found to be heat stable. Competition studies indicated that this binding activity was specific for human lactoferrin. Treatment ofG. vaginalis cells with proteases suggested that this protein was surface exposed. An increase in lactoferrin binding by the 120-kDa protein was observed in G. vaginalis cells grown under iron-restrictive conditions, suggesting that this activity may be iron regulated.