Human RIG-1 ELISA kit

Transcription

1 Human RIG-1 ELISA kit Cat. No.:DEIA6479 Pkg.Size:96T Intended use The RIG-1 (human), ELISA kit is to be used for the quantitative determination of human RIG-1 in cell extracts. General Description The innate immune response to viruses is initiated upon detection of viral RNA generated during the life cycle of most viruses. Recognition of the viral RNA is mediated by either the two cytoplasmic RNA helicases Retinoic acid Inducible Gene-1 (RIG-I ; Ddx58) and Melanoma Differentiation Associated gene 5 (MDA5 / Helicard) or by Toll-like Receptor 3 (TLR3) located to an endosomal-like compartment. RIG-I and MDA5 belong to the family of RIG-I like receptor (RLR) together with the protein LGP2 whose function is still elusive. Upon binding to RNA, RIG-I and MDA5 bind a new mediator named Cardif, MAVS, IPS-1 or VISA that signals through transcription factors, such as IRF-3, IRF-7, and NF-B leading to the activation of interferons (IFNs) and other inflammatory cytokine genes. RIG-I is activated by dsrna and 5 -ppp RNA. RIG-I has been shown to trigger innate immune signaling pathways during infection by orthomyxoviruses, rhabdovirus, vesicular stomatitis virus, and paramyxoviruses; and possibly by the filovirus Ebola virus and by the Epstein-Barr virus. Virus infection induces a rapid production of IFN-/β that leads to expression of hundreds of interferon-stimulated genes (ISGs) whose products direct antiviral actions. RIG-I belongs to the ISG gene family and monitoring its levels using the RIG-I (human) Detection Set (IC) can be used as indicator of IFN production and virus infection. Principle Of The Test This assay is a sandwich Enzyme-Linked Immuno-Sorbent Assay (ELISA) developed for the direct measurement of human RIG-1 (hrig-1) in cell extracts. A monoclonal antibody specific for hrig-1 is coated onto the wells of a microtiter plate. Samples and standards of hrig-1 are pipetted into the wells for binding to the coated antibody. After extensive washing to remove unbound compounds, hrig-1 is recognized by the addition of a biotinylated polyclonal antibody specific for hrig-1. After removal of excess biotinylated antibody, streptavidin-peroxidase is added. Following a final washing, peroxidase activity is quantified using the substrate 3,3,5,5 -tetramethylbenzidine (TMB). The intensity of the color reaction is measured at 450 nm after acidification and is directly proportional to the concentration of hrig-1 in the samples. Reagents And Materials Provided 1. 1 vials human RIG-1 Standard (lyophilized) (1 μg) (STD) 2. 1 vial Detection Antibody (20 μl) (AB) 3. 1 vial Streptavidin HRP (lyophilized) (12.5 μg) (SA-HRP) 4. 1 bottles Wash Buffer 20X (1 x 30 ml) (Wash Buffer 10X) 5. 1 bottle ELISA Buffer 1X (1 x 30 ml) (ELISA Buffer) 6. 1 bottle Lysis Buffer 5 X (1 x 30 ml) (Lysis Buffer 5 x) 7. 1 bottle TMB Substrate Solution (1 x 10 ml) (TMB) 8. 1 bottle Stop Solution (1 x 10 ml) (STOP) 9. 1 Plate coated with RIG-1 Antibody (1 x 96-well strips) 10.2 Plate Covers (plastic film) 1

2 Materials Required But Not Supplied Calibrated precision pipettes Deionized or distilled water Plate washer: automated or manual Plastic tubes for diluting and mixing standard Microtiterplate reader at 450 nm. Specimen Collection And Handling All components of this kit, except the Standard and Detection Antibody, are stable at 4C until the kit s expiration date. The Standard and Detection Antibody must be stored at or below - 20 upon receipt. Avoid freeze/thaw cycles. Plate and reagents should be at room temperature before use. Reagents with a volume less than 100µl should be centrifuged. Sample Preparation. Wash cells with PBS Lyse them in 1X Lysis buffer at a concentration of ~107 cells/ml. Vortex the lysate briefly and incubate for 15 minutes at 4 C (the lysate at that step can be stored at -20 C). Spin at 10,000 rpm for 5 minutes and transfer the supernatant to a new tube. As a start, dilute 1/5 and 1/10 cell extracts in the Lysis buffer (10-50µg of cell extracts /well is suggested). Reagent Preparation Prepare just the appropriate amount of the buffers necessary for the assay! a. The Wash Buffer 20X has to be diluted with deionized or distilled water 1:20 before use (eg 25 ml Wash Buffer 20X ml water) to obtain Wash Buffer 1X. b. The Lysis Buffer 5x is to be diluted with deionized or distilled water 1:5 before use (eg 10 ml Lysis Buffer 5X + 40 ml water) to obtain 1X Lysis Buffer c. The human RIG-1 Standard (STD) has to be reconstituted with 100 µl deionized water. This reconstitution produces a stock solution of 10 µg/ml. Mix the standard to ensure complete reconstitution and allow the standard to sit for a minimum of 15 minutes. Mix well prior to making dilutions.! The reconstituted standard is to be divided and stored at 20 C! Avoid freeze/thaw cycles. Dilute the standard protein concentrate (STD) (10 µg/ml) in 1X Lysis buffer. A seven-point standard curve using 2-fold serial dilutions in 1X Lysis buffer is recommended. Suggested standard points are 10 ng/ml, 5 ng/ml, 2.5 ng/ml, 1.25 ng/ml, ng/ml, ng/ml, ng/ml and 0 ng/ml. Start with the dilution of the concentrate (STD). Table 1. 2

3 Dilute further for the standard curve. Table 2. d. Detection Antibody (AB) has to be diluted 1:1000 in ELISA Buffer(2 µl AB + 2 ml ELISA Buffer). The diluted Detection Antibody is not stable and cannot be stored! e. Streptavidin HRP (SA-HRP) has to be reconstituted with 250 µl deionized water. The reconstituted SA-HRP is stable for one month at 2-8 C! Dilute the reconstituted SA-HRP to the working concentration by adding 10 µl in 10 ml of ELISA Buffer (1:1000). The diluted STREP-HRP is not stable and cannot be stored! Assay Steps 1. Determine the number of plate strips needed for the assay and insert them in the frame for current use. Store the extra strips in the foil bag with desiccant and store at 4 C. 2. Add 300 µl of Wash Buffer (Wash Buffer 1X) using a multichannel pipette or autowasher. Repeat the process for a total of four washes. After the last wash, complete removal of liquid is essential for good performance. 3. Add 100 µl of the different standards (see 9.1. Preparation and Storage of Reagents, section c. human RIG-1 Standard (STD)) into the appropriate wells in duplicate. At the same time, add 100 µl of diluted serum or cell culture supernatant samples in duplicate to the wells. 4. Cover the plate with plastic film and incubate for 3 hours at room temperature. 5. Aspirate the coated wells and add 300 µl of Wash Buffer (Wash Buffer 1X) using a multichannel pipette or autowasher. Repeat the process for a total of four washes. After the last wash, complete removal of liquid is essential for good performance. 3

4 6. Add 100 µl to each well of the diluted Detection Antibody 7. Cover the plate with plastic film and incubate for 1 hour at RT C. 8. Aspirate the coated wells and add 300 µl of Wash Buffer (Wash Buffer 1X) using a multichannel pipette or autowasher. Repeat the process for a total of four washes. After the last wash, complete removal of liquid is essential for good performance. 9. Add 100 µl to each well of the diluted Streptavidin HRP 10. Cover the plate with plastic film and incubate for 30 min at RT C. 11. Aspirate the coated wells and add 300 µl of Wash Buffer (Wash Buffer 1X) using a multichannel pipette or autowasher. Repeat the process for a total of four washes. After the last wash, complete removal of liquid is essential for good performance. 12. Add 100 µl to each well of TMB substrate solution (TMB). 13. Allow the color reaction to develop at RT C in the dark for 5-30 minutes. Do not cover the plate. 14. Stop the reaction by adding 50 µl of Stop Solution (STOP). Tap the plate gently to ensure thorough mixing. The substrate reaction yields a blue solution that turns yellow when Stop Solution (STOP) is added. CAUTION: CORROSIVE SOLUTION! 15. Measure the OD at 450 nm in an ELISA reader. Calculation Average the duplicate readings for each standard, control and sample and subtract the average blank value (obtained with the 0 ng/ml point). Generate the standard curve by plotting the average absorbance obtained for each standard concentration on the vertical (Y) axis vs. the corresponding hrig-1 concentration (ng/ml) on the horizontal axis. Calculate results using graph paper or curve-fitting statistical software. The amount of human RIG-1 in each sample is determined by interpolating for the absorbance value (Y axis) using the standard curve. If the test sample was diluted, multiply the interpolated value by the dilution factor to calculate ng/ml of human RIG-1 in the sample. Typical Standard Curve Typical Standard Curve Figure 1: 4

5 Detection Range ng/ml 10 ng/ml Sensitivity The lowest level of hrig-1 that can be detected by this assay is ng /ml Specificity The antibodies used in this ELISA are specific for the measurement of natural and recombinant human RIG-1. They do not cross - react with mouse RIG-1. Limitations Do not combine leftover reagents with those reserved for additional wells. It is recommended that all standards, controls and samples be run in duplicate. Samples that are > 5 ng /ml should be diluted with 1 X Lysis Buffer. Residual wash liquid should be drained from the wells after last wash by tapping the plate forcefully on absorbent paper. Crystals could appear in the concentrated solutions due to high salt concentration in the stock solutions. Crystals are readily dissolved at room temperature or at 37 C before dilution of the buffer solutions. Once reagents have been added to the plate strips, DO NOT let the strips DRY at any time during the assay. 5

6 The Stop Solution (STOP) consists of hydrochloric acid. Although diluted, the Stop Solution should be handled with gloves, eye protection and protective clothing. Analyte Gene Information Gene Name DDX58 DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 [ Homo sapiens ] Official Symbol Synonyms DDX58 DDX58; DEAD (Asp-Glu-Ala-Asp) box polypeptide 58; probable ATP-dependent RNA helicase DDX58; DKFZp434J1111; FLJ13599; retinoic acid inducible gene I; RIG I; RNA helicase RIG I; RIG-1; RNA helicase RIG-I; DEAD box protein 58; retinoic acid-inducible gene 1 protein; retinoic acidinducible gene I protein; DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide; RIG-I; DKFZp686N19181; GeneID mrna Refseq Protein Refseq NM_ NP_ MIM UniProt ID O95786 Chromosome Location 9p12 Pathway Function REFERENCES Antiviral mechanism by IFN-stimulated genes, organism-specific biosystem; Cytokine Signaling in Immune system, organism-specific biosystem; Cytosolic DNA-sensing pathway, organism-specific biosystem; Cytosolic DNA-sensing pathway, conserved biosystem; Hepatitis C, organism-specific biosystem; Hepatitis C, conserved biosystem; Herpes simplex infection, organism-specific biosystem; ATP binding; ATP-dependent helicase activity; double-stranded DNA binding; double-stranded RNA binding; helicase activity; hydrolase activity, acting on acid anhydrides; identical protein binding; metal ion binding; nucleotide binding; protein binding; zinc ion binding; 1. Takeuchi, O., and Akira, S. (2007). Recognition of viruses by innate immunity. Immun. Rev. 220, Takeuchi, O., and Akira, S. (2008). MDA5/RIG-I and virus recognition. Curr. Opin. Immunol. 20,