NAMPT ELISA Kit

Catalog No. ABIN456795

Product Details NAMPT ELISA Kit

Target: NAMPT (Nicotinamide phosphoribosyltransferase (NAMPT))

Reactivity: Mouse

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 0.312-20.00 ng/mL

Minimum Detection Limit: 0.312 ng/mL

Application: ELISA

Purpose: This immunoassay kit allows for the specific measurement of Mouse visfatin 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 Mouse visfatin.

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

Characteristics: Mus musculus,Mouse,Nicotinamide phosphoribosyltransferase,NAmPRTase,Nampt,Pre-B-cell colony-enhancing factor 1 homolog,PBEF,Visfatin,Nampt,Pbef1,2.4.2.12

Components: Reagent (Quantity): Assay plate (1), Standard (2), Sample Diluent (1x20ml), Assay Diluent A (1x10ml), Assay Diluent B (1x10ml), Detection Reagent A 1×120µl Detection Reagent B 1×120µl 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 visfatin has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any visfatin present is bound by the immobilized antibody. An enzyme-linked polyclonal antibody specific for visfatin 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 visfatin 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 3 produces a stock solution of 32 ng/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The diluted standard serves as the high standard (16 ng/mL). The Sample Diluent serves as the zero standard (0 ng/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. 4
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 visfatin 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 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 NAMPT

Target: NAMPT (Nicotinamide phosphoribosyltransferase (NAMPT))

Alternative Name: Nampt (NAMPT Products)

Synonyms: 1110035O14Rik ELISA Kit, PBEF ELISA Kit, PBEF1 ELISA Kit, VF ELISA Kit, VISFATIN ELISA Kit, AI314458 ELISA Kit, AI480535 ELISA Kit, NAmPRTase ELISA Kit, Pbef ELISA Kit, Pbef1 ELISA Kit, Visfatin ELISA Kit, visfatin ELISA Kit, pbef ELISA Kit, pbef1 ELISA Kit, nicotinamide phosphoribosyltransferase ELISA Kit, Nicotinamide phosphoribosyltransferase ELISA Kit, nicotinamide phosphoribosyltransferase S homeolog ELISA Kit, NAMPT ELISA Kit, Nampt ELISA Kit, nampt ELISA Kit, Smon_0680 ELISA Kit, Mrub_2845 ELISA Kit, Cseg_3704 ELISA Kit, BC1002_3997 ELISA Kit, Ftrac_2686 ELISA Kit, Varpa_5681 ELISA Kit, Celal_3900 ELISA Kit, Deima_0880 ELISA Kit, BC1001_3724 ELISA Kit, Celly_1696 ELISA Kit, Clole_1542 ELISA Kit, Tsp_05934 ELISA Kit, nampt.S ELISA Kit

Background: Visfatin, an adipocytokine that is highly enriched in the visceral fat of both Mouses and mice and whose expression level in plasma increases during the development of obesity. Visfatin corresponds to pre-B cell colony-enhancing factor (PBEF), a 52-kD cytokine expressed in lymphocytes. The gene encoding PBEF was originally isolated from an activated lymphocyte cDNA library.The 3-prime UTR of PBEF contains several TATT motifs, destabilizing sequences characteristic of cytokine messages. The deduced 473-amino acid protein has a calculated molecular mass of 52 kDa. PBEF has a hydrophobic N terminus, 2 N-glycosylation sites, several putative phosphorylation sites, and 6 cysteines. Northern blot analysis detected transcripts of about 2.0, 2.4, and 4.0 kb in all tissues examined, with highest expression in liver, followed by muscle. Although PBEF lacks a typical signal sequence for secretion, transfected COS-7 and mouse embryonic fibroblasts secreted PBEF into the culture medium. Samal et al. found that recombinant PBEF secreted from transfected COS-7 and mouse embryonic fibroblasts was not itself active in a pre-B-cell colony formation assay, but it synergized the pre-B-cell colony formation activity of stem cell factor and interleukin-7. Jia et al. found that PBEF is an inflammatory cytokine that plays a requisite role in the delayed neutrophil apoptosis of sepsis. Visfatin exerted insulin-mimetic effects in cultured cells and lowered plasma glucose levels in mice. Mice heterozygous for a targeted mutation in the visfatin gene had modestly higher levels of plasma glucose relative to wild type littermates. Surprisingly, it was found that visfatin binds to and activates the insulin receptor . Recently, Chen et al. found that plasma level of visfatin in patients with type 2 diabetes mellitus was elevated , suggesting that measurement of plasma visfatin provides a relevant tool for understanding metabolic diseases.