NOS2 ELISA Kit

Catalog No. ABIN455419

Product Details NOS2 ELISA Kit

Target: NOS2 (Nitric Oxide Synthase 2, Inducible (NOS2))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 15.6-1000 pg/mL

Minimum Detection Limit: 15.6 pg/mL

Application: ELISA

Purpose: This immunoassay kit allows for the specific measurement of Human iNOS concentrations in cell culture supernates, serum, and plasma.

Sample Type: Cell Culture Supernatant, Serum, Plasma

Analytical Method: Quantitative

Specificity: This assay recognizes recombinant and natural Human iNOS.

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

Characteristics: Homo sapiens,Human,Nitric oxide synthase, inducible,Hepatocyte NOS,HEP-NOS,Inducible NO synthase,Inducible NOS,iNOS,NOS type II,Peptidyl-cysteine S-nitrosylase NOS2,NOS2,NOS2A,1.14.13.39

Components: Reagent (Quantity): Assay plate (1), Standard (2), Sample Diluent (1x20ml), Assay Diluent A (1x10ml), Assay Diluent B (1x10ml), Detection Reagent A (1x120µl), Detection Reagent B (1x120µl), Wash Buffer(25 x concentrate) (1x30ml), Substrate (1x10ml), Stop Solution (1x10ml)

Application Details

Sample Volume: 100 μL

Plate: Pre-coated

Protocol: This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for iNOS has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any iNOS present is bound by the immobilized antibody. An enzyme-linked polyclonal antibody specific for iNOS 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 iNOS 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 80 U/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 (80 U/mL). The Sample Diluent serves as the zero standard (0 U/mL). 3 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. 4

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 iNOS 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. Note:1 U/mL of partially purified Human iNOS is approximately equal to 1 ng/mL.

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 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 NOS2

Target: NOS2 (Nitric Oxide Synthase 2, Inducible (NOS2))

Alternative Name: NOS2 (NOS2 Products)

Synonyms: HEP-NOS ELISA Kit, INOS ELISA Kit, NOS ELISA Kit, NOS2A ELISA Kit, NOS-II ELISA Kit, Nos-2 ELISA Kit, Nos2a ELISA Kit, i-NOS ELISA Kit, iNOS ELISA Kit, iNos ELISA Kit, NOS2a ELISA Kit, NOS2 ELISA Kit, BmNOS2 ELISA Kit, Nsl ELISA Kit, nos2 ELISA Kit, nitric oxide synthase 2 ELISA Kit, nitric oxide synthase 2, inducible ELISA Kit, inducible nitric oxide synthase ELISA Kit, inducible nitric-oxide synthase ELISA Kit, nanos C2HC-type zinc finger 2 ELISA Kit, NOS2 ELISA Kit, Nos2 ELISA Kit, nos2 ELISA Kit, LOC396821 ELISA Kit, NANOS2 ELISA Kit

Background: NO is produced by a group of enzymes called nitric oxide synthases (NOS). These enzymes catalyze the production of NO and L-citrulline from L-arginine, O2, and NADPH-derived electrons. Mammalian systems contain three well-characterized isoforms of the enzyme: neuronal NOS (nNOS, also called NOS1), inducible NOS (iNOS or NOS2), and endothelial NOS (eNOS or NOS3). The names reflect characteristics of the activity or the original tissues in which the enzymes were first described, but it is now known that each of these isoforms is expressed in a variety of tissues and cell types . The three main isoforms share structural similarities and have nearly identical catalytic mechanisms. They all require a number of cofactors and prosthetic groups for activity including FAD, FMN,heme, calmodulin, and tetrahydrobiopterin. The homodimeric form is required for NO production, and the subunits have molecular masses of approximately 160 kDa (nNOS), 135 kDa (eNOS), and 130 kDa (iNOS). Three distinct domains are necessary for catalytic activity. Starting at the C-terminus there is a reductase domain, a calmodulin-binding domain,and an oxygenase domain. The reductase domain contains the FAD and FMN moieties and shares extensive amino acid (aa) homology with cytochrome P-450 reductase. This domain transfers electrons from NADPH to the oxygenase domain. The oxygenase domain actually catalyzes the conversion of arginine into citrulline and NO and contains the binding sites for heme, tetrahydrobiopterin, and arginine. Calmodulin binding is required for activity of all of the NOS isoforms.

Pathways: Retinoic Acid Receptor Signaling Pathway, Cellular Response to Molecule of Bacterial Origin, Inositol Metabolic Process, Regulation of Leukocyte Mediated Immunity, Positive Regulation of Immune Effector Process