Objectives: Anti-TIF1γ is an important autoantibody in the diagnosis of cancer-associated dermatomyositis and the most common autoantibody in juvenile onset dermatomyositis. Its reliable detection is important to instigate further investigations into underlying malignancy in adults. We previously showed that commercial assays using line and dot blots do not reliably detect anti-TIF1γ. We aimed to test a new commercial ELISA and compare with previously obtained protein immunoprecipitation.
Methods: Radio-labelled immunoprecipitation had previously been used to determine the autoantibody status of patients with immune-mediated inflammatory myopathies and several healthy controls. ELISA was undertaken on healthy control and anti-TIF1γ sera and compared to previous immunoprecipitation data.
Results: A total of 110 serum samples were analysed: 42 myositis patients with anti- TIF1γ and 68 autoantibody negative healthy control sera. Anti-TIF1γ was detected by ELISA in 41 out of 42 of the anti-TIF1γ-positive samples by immunoprecipitation, and in none of the healthy controls, giving a sensitivity of 97.6% and specificity of 100%. The false negative rate was 2%.
Conclusion: ELISA is an affordable and time-efficient method which is accurate in detecting anti-TIF1γ.
Affiblot: a dot blot-based screening device for selection of reliable antibodies
The key factor in the development of antibody-based assays is to find an antibody that has an appropriate affinity, high specificity, and low cross-reactivity. However, this task is not easy to carry out since the research antibodies on the market may suffer from low specificity and reproducibility. Here, we report on a palm-sized dot blot-based device, called the affiblot, that has a specially designed lid that allows simultaneous semi-quantitative comparison of up to five antibodies from different suppliers regarding their affinity/avidity, cross-reactivity, and batch-to-batch reliability.
The only required peripheral equipment is a vacuum pump, a camera, and densitometry software. The affiblot device was tested for its functionality and its measurements were compared against those obtained by standard dot blot and ELISA. The benefit over these methods, when various antibodies are evaluated, is in its simplicity. It allows easy antigen deposition, fast application and the discarding of the solutions, a compact undivided membrane, and therefore significant decrease of labor. The device was tested with specific anti-ApoE, anti-EpCAM, anti-Salmonella, anti-E. coli, and anti-Listeria antibodies from different suppliers. Their properties were compared for their ability to interact specifically with antigen and/or non-target structures and the best-suited antibody for the intended application was identified.
Comparative evaluation of indirect-ELISA and DOT blot assay for serodetection of Mycoplasma gallisepticum and Mycoplasma synoviae antibodies in poultry
Avian mycoplasmosis, mainly caused by Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) is an economically important disease of the poultry industry. The present study was aimed to develop whole cell based indirect-ELISA (i-ELISA) and DOT blot assay (DOT-ELISA) as rapid, sensitive, specific and economical sero-detection tests for MG and MS. A total of 306 blood samples were collected from birds slaughtered at local meat shops of different districts of Haryana, India to detect MG and MS antibodies. Sonicated antigens prepared from freshly grown culture of MG and MS were used to develop i-ELISA and DOT blot assay.
In i-ELISA, 50.32% and 61.76% serum samples were found to be positive for MG and MS antibodies, respectively. However in DOT blot assay, 41.83% and 53.92% serum samples were found positive for MG and MS antibodies, respectively. The relative diagnostic sensitivity and specificity of DOT-ELISA were measured considering i-ELISA as a reference test. The relative diagnostic sensitivity of the DOT blot assay was found to be 69.48% and 82.01%; whereas relative diagnostic specificity was 86.18% and 91.45% for the detection of MG and MS antibodies, respectively. The developed serological assays may be used as rapid and economical diagnostic tools for large scale screening of poultry sera for MG and MS antibodies.
Multiple antigenic peptide-based flow through dot–blot assay for simultaneous antibody detection of infectious bronchitis virus and Newcastle disease virus
The present study describes the development of a novel affordable and rapid visual dot-blot assay using synthetic multiple antigenic peptides (MAP) for simultaneous detection of antibodies to infectious bronchitis virus (IBV) and Newcastle disease virus (NDV). Antibody detection efficiencies of MAP peptides namely, NP1 MAP (Nucleoprotein IBV) and HN MAP (Haemagglutinin-neuraminidase NDV) were studied in solid-phase indirect peptide ELISA. In comparison with the commercial kit, the NP1 MAP showed 89.20% diagnostic sensitivity (DSn) and 85.90% diagnostic specificity (DSp) at 19.45% ROC cut-off.
Similarly, HN MAP was evaluated and showed 89.70% DSn and 92.90% DSp at 19.90 % ROC cut-off. The peptides after evaluating their ELISA performance were further used to device a flow-through dot-blot assay (FT-DBA) for simultaneous detection of IBV and NDV antibodies. The kappa value for IBV by FT-DBA in comparison to commercial ELISA was 0.64 whereas for NDV, FT-DBA gave a kappa value of 0.68 in comparison to commercial ELISA indicating substantial agreement between the assays. In essence, the divergent MAP based diagnostic design could provide an alternative for antibody detection of IBV and NDV. Further, the FT-DBA approach could be used for low cost, rapid and pen-side detection of IBV and NDV antibodies simultaneously.
Dot blots of solubilized extracellular matrix allow quantification of human antibodies bound to epitopes present in decellularized porcine pulmonary heart valves
Background: The present study reports the development of a sensitive dot blot protocol for determining the level of preformed antibodies against porcine heart valve tissue derived from wild-type (WT) and α-Gal-KO (GGTA1-KO) pigs in human sera.
Methods: The assay uses decellularized and solubilized heart valve tissue; antibody binding found in this dot blot assay could be correlated with antibody titers of preformed anti-α-Gal and anti-Neu5Gc antibodies detected by a sensitive ELISA.
Results: The ultimate protocol had an inter-assay variance of 9.5% and an intra-assay variance of 9.2%, showing that the test is reliable and highly reproducible. With the aid of this dot blot assay, we found significant variation with regard to antibody contents among twelve human sera. Binding of preformed antibodies to WT tissue was significantly higher than to GGTA1-KO tissue.
Conclusions: The dot blot assay described herein could be a valuable tool to measure preformed antibody levels in human sera against unknown epitopes on decellularized tissue prior to implantation. Ultimately, this prescreening may allow a matching of the porcine xenograft with the respective human recipients in demand and thus may become an important tool for graft long-term survival similar to current allotransplantation settings.
Western blot Kit for Rat Primary Antibodies, Chemilum. Substrate |
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| 80203-Rt | Alpha Diagnostics | 1 kit | 781.2 EUR |
Western blot Kit for Pig Primary Antibodies; Chemilum. Substrate |
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| 80209-Pg | Alpha Diagnostics | 1 kit | 781.2 EUR |
Western blot Kit for Goat Primary Antibodies, Chemilum. Substrate |
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| 80201-Gt | Alpha Diagnostics | 1 kit | 781.2 EUR |
Western blot Kit for Mouse Primary Antibodies, Chemilum. Substrate |
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| 80202-Mo | Alpha Diagnostics | 1 kit | 781.2 EUR |
Western blot Kit for Human Primary Antibodies, Chemilum. Substrate |
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| 80207-Hu | Alpha Diagnostics | 1 kit | 781.2 EUR |
Western blot Kit for Sheep Primary Antibodies; Chemilum. Substrate |
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| 80208-Sh | Alpha Diagnostics | 1 kit | 781.2 EUR |
Western blot Kit for Rabbit Primary Antibodies, Chemilum. Substrate |
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| 80200-Rb | Alpha Diagnostics | 1 kit | 781.2 EUR |
Western blot Kit for Monkey Primary Antibodies, Chemilum. Substrate |
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| 80205-Mk | Alpha Diagnostics | 1 kit | 781.2 EUR |
Western blot Kit for Chicken Primary Antibodies, Chemilum. Substrate |
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| 80204-Ch | Alpha Diagnostics | 1 kit | 781.2 EUR |
Western blot Kit for G. pig Primary Antibodies, Chemilum. Substrate |
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| 80206-Gp | Alpha Diagnostics | 1 kit | 781.2 EUR |
LL37 (Human) - Dot Blot Kit |
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| DBK-075-06 | PHOENIX PEPTIDE | 5 blots | 805.68 EUR |
Blocking Buffer and Antibody Diluent for Dot Blot and Western Blot Kits |
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| WB-B003 | PHOENIX PEPTIDE | 50 ml | 66.96 EUR |
Dot blot manifold 48 well - EACH |
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| CSLD48 | Scientific Laboratory Supplies | EACH | 793.8 EUR |
Dot blot manifold 96 well - EACH |
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| CSLD96 | Scientific Laboratory Supplies | EACH | 932.85 EUR |
Dot blot manifold 24 well - EACH |
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| CSLS24 | Scientific Laboratory Supplies | EACH | 932.85 EUR |
Ghrelin (Rat, Mouse) - Dot Blot Kit |
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| DBK-031-31 | PHOENIX PEPTIDE | 5 blots | 805.68 EUR |
Neuromedin U (Rat) - Dot Blot Kit |
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| DBK-046-41 | PHOENIX PEPTIDE | 5 blots | 805.68 EUR |
TBS, pH7.6 - Buffer for Dot Blot and Western Blot Kits |
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| WB-B002 | PHOENIX PEPTIDE | 50 ml | 18.36 EUR |
TBST, pH7.6 - Buffer for Dot Blot and Western Blot Kits |
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| WB-B001 | PHOENIX PEPTIDE | 100 ml | 25.92 EUR |
Detection Reagent A for Dot Blot and Western Blot Kits |
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| WB-B006 | PHOENIX PEPTIDE | 30 ml | 86.4 EUR |
Detection Reagent B for Dot Blot and Western Blot Kits |
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| WB-B007 | PHOENIX PEPTIDE | 30 ml | 86.4 EUR |
Oxidative Stress Antibodies Set | 5 antibodies |
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| AS19-4294 | Agrisera AB | each | 1472 EUR |
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.
This study evaluated the specificity of different avian secondary antibodies used in Western blot and dot-blot ELISA to detect avian bornavirus antibodies in bird plasma.Plasma samples were collected from: two Blue and gold macaws, one positive and one negative for avian bornavirus by RT-PCR; a Cockatiel and a Monk parakeet prior to and following experimental infection; and, two Mallards, one positive and one negative for avian bornavirus by RT-PCR Samples were analyzed by Western blot and dot-blot ELISA that incorporated recombinant avian bornavirus nucleoprotein as the target analyte. Four species-specific anti-IgY secondary antibodies were used in the assays: goat anti-macaw IgY, goat anti-bird IgY, goat anti-duck IgY, and rabbit anti-chicken IgY.
Results
In the Western blot, anti-macaw IgY secondary antibody produced strong signals with Blue and gold macaw and Cockatiel positive plasma, but no signal with Mallard positive plasma. Anti-bird IgY secondary antibody produced strong signals with Blue and gold macaw, Cockatiel, and Mallard positive plasma. Anti-duck and anti-chicken IgY secondary antibody produced a strong and moderate signal, respectively, only with Mallard positive plasma. In the dot-blot ELISA, there was a distinct and significant difference (P<0.05) in the signal intensity between the different secondary antibodies within a bird species. Anti-macaw IgY secondary antibody produced significantly (P<0.05) stronger signals than the other secondary antibodies in Blue and gold macaw, Cockatiel, and Monk parakeet positive plasma, while anti-duck IgY secondary antibody produced significantly (P<0.05) stronger signals than the other secondary antibodies in Mallard positive plasma.In testing psittacines with immunoassays, and especially in assays that incorporate short incubation reaction times such as a dot-blot ELISA, species-specific anti-IgY secondary antibodies provided more accurate results.