IL1RN ELISA Kit

Catalog No. ABIN579088

Product Details IL1RN ELISA Kit

Target: IL1RN (Interleukin 1 Receptor Antagonist (IL1RN))

Reactivity: Pig

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 0.156-10 ng/mL

Minimum Detection Limit: 0.156 ng/mL

Application: ELISA

Purpose: This immunoassay kit allows for the specific measurement of porcine Interleukin 1 receptor1, IL-1ra concentrations in cell culture supernates, serum and plasma.

Sample Type: Cell Culture Supernatant, Serum, Plasma

Analytical Method: Quantitative

Specificity: 4 This assay recognizes recombinant and natural porcine IL-1ra.

Cross-Reactivity (Details): No significant cross-reactivity or interference was observed.

Sensitivity: The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest detectable concentration that could be differentiated from zero.

Characteristics: Sus scrofa,Pig,Interleukin-1 receptor antagonist protein,IL-1RN,IL-1ra,IRAP,IL1 inhibitor,IL1RN,IRAP1

Components: Reagent (Quantity ): Assay plate (1), Standard (2), Sample Diluent (1 × 20ml), Assay Diluent A (1x10ml), Assay Diluent B (1x10ml), Detection Reagent A (1 × 120μl), Detection Reagent B (1 × 120μl), Wash Buffer (25 x concentrate) (1 × 30ml), Substrate (1x10ml), Stop Solution (1 x 10ml)

Application Details

Sample Volume: 100 μL

Plate: Pre-coated

Protocol: This assay employs the quantitative sandwich enzyme immunoassay technique. A antibody specific for IL-1ra has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IL-1ra present is bound by the immobilized antibody. An enzyme-linked antibody specific for IL-1ra is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of IL-1ra bound in the initial step. The color development is stopped and the intensity of the color is measured.

Reagent Preparation:

Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 500 ml of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 3,000 pg/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard (3,000 pg/mL). The Sample Diluent serves as the zero standard (0 pg/mL). Detection Reagent A and B - Dilute to the working concentration specified on the vial label using Assay Diluent A and B (1:100), respectively.

Sample Collection: Cell culture supernates - Remove particulates by centrifugation and assay immediately or aliquot and store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20 °C. Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 x g at 2 - 8 °C within 30 minutes of collection. Store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Note: Citrate plasma has not been validated for use in this assay.

Assay Procedure:

Allow all reagents to reach room temperature. Arrange and label required number of strips.
1. Prepare all reagents, working standards and samples as directed in the previous sections.
2. Add 100 uL of Standard, Control, or sample* per well. Cover with the adhesive strip. Incubate for 2 hours at 37 °C.
3. Remove the liquid of each well, don’t wash.
4. Add 100 uL of Detection Reagent A to each well. Incubate for 1 hour at 37°C. Detection Reagent A may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
5. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (350 uL) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
6. Add 100 uL of Detection Reagent B to each well. Cover with a new adhesive strip.Incubate for 1 hours at 37 °C.
7. Repeat the aspiration/wash as in step
5. 8. Add 90 uL of Substrate Solution to each well. Incubate for 30 minutes at room temperature. Protect from light.
9. Add 50 uL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
10. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.
Important Note:1. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
2. It is recommended that no more than 32 wells be used for each assay run if manual pipetting is used since pipetting of all standards, specimens and controls should be completed within 5 minutes. A full plate of 96 wells may be used if automated pipetting is available.
3. Duplication of all standards and specimens, although not required, is recommended.
4. When mixing or reconstituting protein solutions, always avoid foaming.
5. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
6. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.

Calculation of Results:

Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the IL-1ra concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

Restrictions: For Research Use only

Handling

Handling Advice: 1. The kit should not be used beyond the expiration date on the kit label.
2. Do not mix or substitute reagents with those from other lots or sources.
3. If samples generate values higher than the highest standard, further dilute the samples with the Assay Diluent and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause 3 variation in binding.
4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.

Storage: 4 °C/-20 °C

Storage Comment: The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.

Target Details for IL1RN

Target: IL1RN (Interleukin 1 Receptor Antagonist (IL1RN))

Alternative Name: IL1RN (IL1RN Products)

Synonyms: DIRA ELISA Kit, ICIL-1RA ELISA Kit, IL-1RN ELISA Kit, IL-1ra ELISA Kit, IL-1ra3 ELISA Kit, IL1F3 ELISA Kit, IL1RA ELISA Kit, IRAP ELISA Kit, MVCD4 ELISA Kit, IRAP1 ELISA Kit, IL1RN ELISA Kit, IL-1RA ELISA Kit, F630041P17Rik ELISA Kit, interleukin 1 receptor antagonist ELISA Kit, interleukin 1 receptor antagonist-like ELISA Kit, IL1RN ELISA Kit, Il1rn ELISA Kit, IL1RNL ELISA Kit

Background: The two forms of Interleukin-1 (IL-1alphaand IL-1beta) are cytokines produced by mononuclear phagocytes, skin keratinocytes, some epithelial cells, and some cells of the CNS. IL-1alphaand IL-1beta are thus important mediators of the inflammatory and immune responses of animals. They play important roles in the production of pathological conditions resulting in chronic inflammation, septic shock, and defects in hematopoiesis. The effects produced by the IL-1s result from the binding of these factors to two distinct cell surface receptors, IL-1 R Types I and II. Type I receptor is an 80 kDa protein found on T cells, fibroblasts, and keratinocytes. Type II receptor is a 68 kDa protein found on B cells and PMNs. In general, the Type I receptor binds to IL-1alphaor IL-1beta with approximately equal affinity and the Type II receptor binds IL-1beta more strongly than IL-1alpha, but only the Type I receptor is capable of transducing a signal and can produce all of the biological effects attributed to IL-1. IL-1 receptor antagonist is a member of the Interleukin 1 cytokine family and a different type of naturally occurring inhibitor of IL-1 activity, it inhibits IL-1alpha and IL-1beta binding to interleukin receptors. A cDNA encoding this polypeptide has been isolated from monocytes and found to code for a mature 152 amino acid residue glycoprotein of 25,000 native molecular weight. This molecule, known as secreted IL-1 receptor antagonist, shows 26% amino acid homology to IL-1betaand 19% homology to IL-1alpha. Evidence indicates that the inhibitory action of sIL-1ra results from binding of IL-1ra to the IL-1 receptor Type I with an affinity comparable to that of IL-1alphaor IL-1beta (Kd ~ 200 pM), thus competing with IL-1alphaor IL-1betafor binding to this receptor. This binding, however, does not result in signal transduction. IL-1ra binds to the IL-1 receptor Type II with considerably lower affinity than that shown by IL-1beta. This makes sense teleologically in that two mechanisms designed to inhibit the actions of IL-1betado not compete with each other. Cells known to produce IL-1ra include monocytes, neutrophils, macrophages and fibroblasts. Cytokines known to upregulate IL-1ra production include IL-13, IL-6, IL-4, IFN-gamma, GM-CSF and TGF-beta, the latter apparently by triggering IL-1 production which itself triggers IL-1ra synthesis. The amino acid sequences of IL-1ra from at least four species have been determined (porcine, rat, mouse and rabbit) and found to be at least 75% 2 homologous. IL-1ra can also be synthesized as a strictly intracellular form whose production is the result of an alternative splicing of exon 1.

Gene ID: 3535

Pathways: NF-kappaB Signaling, Hormone Transport, Cancer Immune Checkpoints