Adenovirus Kit ELISA

N° du produit ABIN2345182

Détail du produit Adenovirus Kit ELISA

Antigène: Adenovirus (HAdV) (Human Adenovirus (HAdV))

Reactivité: Adenovirus

Méthode de détection: Colorimetric

Type de méthode: Sandwich ELISA

Application: ELISA

Marque: QuickTiter™

Type d'échantillon: Cell Extracts

Analytical Method: Quantitative

Attributs du produit: A particular challenge in the delivery of a gene by a viral vector is the accurate measurement of virus titer. Traditionally, infectivity particles are measured in culture by a plaque-forming unit assay (PFU) that scores the number of viral plaques as a function of dilution. These methods are time-consuming (10 days), require a long infection period, and suffer from a high degree of inter-assay variability and are affected by virus-cell interactions. QuickTiter™ Adenovirus Titer ELISA Kit utilizes an antibody against adenovirus hexon proteins to quantitate infected cells. The hexon proteins are the largest and most abundant of the structural proteins in the adenovirus capsid, and they are distributed symmetrically to form capsid facets. QuickTiter™ Adenovirus Titer ELISA Kit provides a quick and complete system to functionally titer virus infectivity, it provides sufficient reagents for up to 192 tests in 96-well plates. In contrast to the 10-day infection of a classical plaque assay, the kit only requires a 2-day infection. Detection sensitivity is 104 ifu/mL (106-107 VP/mL), which is sufficient for most adenoviral samples. The kit recognizes all 41 serotypes of adenovirus by immunocytochemistry and can be used with any adenovirus system as long as the virus is able to amplify in HEK 293 cells.

Ingrédients: Box 1 (shipped at room temperature)

1. Anti-Hexon Antibody : One tube - 30 μL.
2. Secondary Antibody, HRP Conjugate : One tube - 50 μL.
3. TMB Substrate : One amber bottle - 20 mL.
4. Stop Solution : One bottle - 20 mL.

Box 2 (shipped on blue ice packs)

1. Ad-β gal Positive Control : One tube - 50 μL at 1.0 x 109 ifu/mL.

Matériel non inclus:

1. Recombinant adenovirus of interest
2. HEK 293 cells and cell culture growth medium
3. Methanol
4. 1 % BSA/PBS
5. Wash Buffer such as PBS
6. Microtiter plate reader

Information d'application

Commentaires:

• More accurate adenoviral titer than traditional plaque-forming unit assays
• Faster results: 2.5 days vs. 10 days
• No agar overlay steps

Durée du test: 2.5 d

Plaque: Without plate

Préparation des réactifs:

• 1X Anti-Hexon antibody solution: Prepare a 1X anti-hexon antibody solution by diluting the provided Anti-Hexon antibody stock 1:1000 in 1 % BSA/PBS. Store the diluted solution on ice.
• 1X Secondary antibody solution: Prepare a 1X Secondary antibody solution by diluting the provided stock 1:2000 in 1 % BSA/PBS. Store the diluted solution on ice.

Préparation de l'échantillon:

• For unknown viral samples, create 10-fold serial dilutions with culture medium. For example, start with a 1:100 dilution of the original viral samples by adding 10 μL of viral sample to a sterile tube containing 990 μL of culture medium. Transfer 100 μL of the mixture to the next tube containing 900 μL of culture medium. Repeat this step several times. For accurate assessment of viral titer, one of the dilutions for your unknown viral sample should be within the range of the Ad-β gal standard curve (4.0 x 103 ifu/mL to 2.5 x 105 ifu/mL).

Procédure de l'essai:

I. Virus Infection

1. Harvest HEK 293 cells and resuspend cells in culture medium at 5 x 105 cells/mL. Seed 100 μL in each well of a 96-well plate and incubate at 37 °C, 5 % CO2 for 1 hr. Note: Adenovirus titer assay is critically dependent on the firm attachment of cells. If the cells look thin and easy to come off during immunostaining steps, you won't get consistent results. Only use low passage 293 cells with flattened morphology or 293AD (Cat. # AD-100), a selected 293 cell line for plasmid transfection, adenovirus amplification and titering. To improve cell adhesion, you can also precoat the plate with polylysine or extracellular matrix.
2. Prepare serial dilutions of the Ad-β gal positive control and your viral sample in culture medium. Dropwise add 50 μL of diluted viral sample to each well of the 96-well assay plate (note: a negative control should be performed simultaneously). To ensure accuracy, perform each sample in duplicate.
3. Incubate infected cells at 37 °C, 5 % CO2 for 2 days.

II. Immunoassay

1. Slowly remove medium from the wells by tilting the plate and aspirating from the edge, then fix infected 293 cells by gently adding 100 μL of cold methanol down the side of each well of the 96-well assay plate, taking care not to dislodge the cells. Incubate 20 minutes at -20 °C.
2. Gently wash the fixed cells three times with 1X PBS, five minutes each wash.
3. Block for 1 hr with 200 μL of 1 % BSA in PBS per well at room temperature on an orbital shaker.
4. Add 100 μL of diluted 1X anti-Hexon antibody solution to each well and incubate for 1 hr at room temperature on an orbital shaker.
5. Gently wash the fixed cells three times with 1X PBS, five minutes each wash.
6. Add 100 μL of diluted 1X Secondary antibody solution (HRP-conjugated) to each well and incubate for 1 hr at room temperature on an orbital shaker. 4
7. Gently wash the fixed cells five times with 1X PBS, five minutes each wash.
8. Warm TMB Substrate to room temperature. Add 100 μL of TMB Substrate solution and incubate at room temperature for 5 to 10 minutes. Stop reaction by adding 100 μL of Stop Solution to each well.
9. Measure Optical Density at 450nm on a 96-well plate reader. Calculate the viral titer based on the standard curve from Ad-β gal positive control titrations.

Restrictions: For Research Use only

Stockage

Précaution d'utilisation: Remember that you will be working with samples containing infectious virus. Follow the recommended NIH guidelines for all materials containing BSL-2 organims.

Stock: 4 °C/-80 °C

Stockage commentaire: Upon receipt, store the Ad-β gal Positive Control at -80°C. Store all other kit components at 4°C until their expiration dates.

Références pour Adenovirus Kit ELISA (ABIN2345182)

García-Pascual, Martínez, Calvo, Ferrero, Villanueva, Pozuelo-Rubio, Soengas, Tormo, Simón, Pellicer, Gómez: "Evaluation of the potential therapeutic effects of a double-stranded RNA mimic complexed with polycations in an experimental mouse model of endometriosis." dans: Fertility and sterility, Vol. 104, Issue 5, pp. 1310-8, (2015) (PubMed).

Gibson, Munns, Freytag, Barton, Veenstra, Bettahi, Bissonette, Wei: "Immunotherapeutic intervention with oncolytic adenovirus in mouse mammary tumors." dans: Oncoimmunology, Vol. 4, Issue 1, pp. e984523, (2015) (PubMed).

Lakshmanan, Zhang, Nweze, Du, Harbrecht: "Glycogen synthase kinase 3 regulates IL-1? mediated iNOS expression in hepatocytes by down-regulating c-Jun." dans: Journal of cellular biochemistry, Vol. 116, Issue 1, pp. 133-41, (2014) (PubMed).

Oh, Kang, Ooi, Choi, Sage, Rhee: "Overexpression of SPARC in human trabecular meshwork increases intraocular pressure and alters extracellular matrix." dans: Investigative ophthalmology & visual science, Vol. 54, Issue 5, pp. 3309-19, (2013) (PubMed).

Muruganandan, Parlee, Rourke, Ernst, Goralski, Sinal: "Chemerin, a novel peroxisome proliferator-activated receptor gamma (PPARgamma) target gene that promotes mesenchymal stem cell adipogenesis." dans: The Journal of biological chemistry, Vol. 286, Issue 27, pp. 23982-95, (2011) (PubMed).

Hoashi, Tamaki, Hearing: "The secreted form of a melanocyte membrane-bound glycoprotein (Pmel17/gp100) is released by ectodomain shedding." dans: FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Vol. 24, Issue 3, pp. 916-30, (2010) (PubMed).

Tudhope, Catley, Fenwick, Russell, Rumsey, Newton, Barnes, Donnelly: "The role of IkappaB kinase 2, but not activation of NF-kappaB, in the release of CXCR3 ligands from IFN-gamma-stimulated human bronchial epithelial cells." dans: Journal of immunology (Baltimore, Md. : 1950), Vol. 179, Issue 9, pp. 6237-45, (2007) (PubMed).

Détail du antigène

Antigène: Adenovirus (HAdV) (Human Adenovirus (HAdV))

Autre désignation: Adenovirus (HAdV Produits)

Classe de substances: Virus

Sujet: Recombinant adenoviruses have tremendous potential in both research and therapeutic applications. There are numerous advantages they provide when introducing genetic material into host cells. The permissive host cell range is very wide. The virus has been used to infect many mammalian cell types (both replicative and non-replicative) for high expression of the recombinant protein. Recombinant adenoviruses are especially useful for gene transfer and protein expression in cell lines that have low transfection efficiency with liposome. After entering cells, the virus remains epichromosomal (i.e. does not integrate into the host chromosome so does not activate or inactivate host genes). Recently, recombinant adenoviruses have been used to deliver RNAi into cells. HEK 293 cells or their variants are used as host cells for viral amplification. Recombinant adenoviruses can be grown at high titer (1010 VP (viral particles)/mL, which can be concentrated up to 1013 VP/mL) and purified by