MIF ELISA Kit

Catalog No. ABIN455789

Product Details MIF ELISA Kit

Target: MIF (Macrophage Migration Inhibitory Factor (Glycosylation-Inhibiting Factor) (MIF))

Reactivity: Mouse

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 62.5-4000 pg/mL

Minimum Detection Limit: 62.5 pg/mL

Application: ELISA

Purpose: This immunoassay kit allows for the in vitro quantitative determination of mouse MIF concentrations in serum, plasma and other biological fluids.

Sample Type: Plasma, Serum

Analytical Method: Quantitative

Specificity: This assay recognizes recombinant and natural mouse MIF.

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

Characteristics: Mus musculus,Mouse,Macrophage migration inhibitory factor,MIF,Delayed early response protein 6,DER6,Glycosylation-inhibiting factor,GIF,L-dopachrome isomerase,L-dopachrome tautomerase,Phenylpyruvate

Components: Reagent (Quantity):

• Assay plate (1),
• Standard (2),
• Sample Diluent (1×20 mL),
• Assay Diluent A (1×10 mL),
• Assay Diluent B (1×10 mL),
• Detection Reagent A (1×120 μL),
• Detection Reagent B (1×120 μL),
• Wash Buffer(25 x concentrate) (1×30 mL),
• Substrate (1×10 mL),
• 2 Stop Solution (1×10 mL),
• Plate sealer for 96 wells (5),
• Instruction (1)

Material not included: Microplate reader. Pipettes and pipette tips. EP tube Deionized or distilled water.

Application Details

Sample Volume: 100 μL

Plate: Pre-coated

Protocol: The microtiter plate provided in this kit has been pre-coated with an antibody specific to MIF. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for MIF. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. Only those wells that contain MIF, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a 2 sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm 2 nm. The concentration of MIF in the samples is then determined by comparing the O.D. of the samples to the standard curve.

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 30 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 750 mL of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions (Making serial dilution in the wells directly is not permitted). The undiluted standard serves as the high standard. The Sample Diluent serves as the zero standard (0 ng/ml).

Sample Collection: Serum - Use a serum separator tube and allow samples to clot for 30 minutes before centrifugation for 20 minutes at approximately 1000 g. Remove serum and assay immediately or aliquot and store samples at -20 or -80 . Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 g at 2 - 8 within 30 minutes of collection. Store samples at -20 or -80 . Avoid repeated freeze-thaw cycles. Other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20 or -80 . Avoid repeated freeze-thaw cycles. Sample preparation - Serum/plasma samples require a 10-20 fold dilution. Sample should be diluted by 0.02 M PBS(PH=7.0-7.2). Note: Serum and plasma to be used within 7 days may be stored at 2-8 , otherwise samples must stored at -20 ( 1 month) or -80 ( 2 months) to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature.

Assay Procedure:

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37 °C directly.). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at 4 °C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.
1. Add 100 μL of Standard, Blank, or Sample per well. Cover with the Plate sealer. Incubate for 2 hours at 37 °C .
2. Remove the liquid of each well, don ’ t wash.
3. Add 100 μL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Incubate for 1 hour at 37 °C . Detection Reagent A working solution may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
4. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (approximately 400 μL) 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.
5. Add 100 μL of Detection Reagent B working solution to each well. Cover with a new Plate sealer. Incubate for 1 hours at 37 °C .
6. Repeat the aspiration/wash as in step 4.
7. Add 90 μL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate within 30 minutes at 37 °C . Protect from light.
8. Add 50 μL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
9. Determine the optical density of each well at once, using a microplate reader set to 450 nm.
Important Note:
1. Absorbance is a function of the incubation time. Therefore, prior to starting the assay it is recommended that all reagents should be freshly prepared prior to use and all required strip-wells are secured in the microtiter frame. This will ensure equal elapsed time for each pipetting step, without interruption.
2. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals have completely dissolved. The reconstituted Standards can be used only once. This assay requires pipetting of small volumes. To minimize imprecision caused by pipetting, ensure that pipettors are calibrated. It is recommended to suck more than 10 μ l for once pipetting.
3. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Once reagents have been added to the well strips, DO NOT let the 5 strips DRY at any time during the assay.
4. For each step in the procedure, total dispensing time for addition of reagents to the assay plate should not exceed 10 minutes.
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. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
7. Duplication of all standards and specimens, although not required, is recommended.
8. Substrate Solution is easily contaminated. Please protect it from light.

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 x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the SAA concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. It is recommended to use some related software to do this calculation, such as curve expert 13.0. 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. 3
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 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.
5. Limited by the current condition and scientific technology, we can't completely conduct the comprehensive identification and analysis on the raw material provided by suppliers. So there might be some qualitative and technical risks to use the kit.

Storage: 4 °C/-20 °C

Storage Comment: The Assay Plate, Standard, Detection Reagent A and Detection Reagent B should be stored at -20°C upon being received. After receiving the kit , Substrate should be always stored at 4°C.

References for MIF ELISA Kit (ABIN455789)

Sun, Zhang, Zhao, Zhen, Huang, Wang, He, Liu, Xu, Yang, Qu, Ma, Zhang, Zhang, Hu: "Attenuation of atherosclerotic lesions in diabetic apolipoprotein E-deficient mice using gene silencing of macrophage migration inhibitory factor." in: Journal of cellular and molecular medicine, Vol. 19, Issue 4, pp. 836-49, (2016) (PubMed).

White, Fang, Chan, Morand, Kiriazis, Duffy, Taylor, Dart, Du, Gao: "Pro-inflammatory action of MIF in acute myocardial infarction via activation of peripheral blood mononuclear cells." in: PLoS ONE, Vol. 8, Issue 10, pp. e76206, (2014) (PubMed).

Mathew, Jacobson, Siegler, Moitra, Blasco, Xie, Unzueta, Zhou, Evenoski, Al-Sakka, Sharma, Huey, Bulent, Smith, Jayaraman, Reddy, Reddy, Fingerle-Rowson, Bucala, Dudek, Natarajan, Weichselbaum, Garcia: "Role of migratory inhibition factor in age-related susceptibility to radiation lung injury via NF-E2-related factor-2 and antioxidant regulation." in: American journal of respiratory cell and molecular biology, Vol. 49, Issue 2, pp. 269-78, (2013) (PubMed).

Target Details for MIF

Target: MIF (Macrophage Migration Inhibitory Factor (Glycosylation-Inhibiting Factor) (MIF))

Alternative Name: Mif (MIF Products)

Synonyms: mif ELISA Kit, Mif ELISA Kit, gif ELISA Kit, glif ELISA Kit, mmif ELISA Kit, LOC100136498 ELISA Kit, LOC100284350 ELISA Kit, LOC100284546 ELISA Kit, GIF ELISA Kit, Glif ELISA Kit, GLIF ELISA Kit, MMIF ELISA Kit, macrophage migration inhibitory factor L homeolog ELISA Kit, macrophage migration inhibitory factor ELISA Kit, macrophage migration inhibitory factor (glycosylation-inhibiting factor) ELISA Kit, mif.L ELISA Kit, mif ELISA Kit, MIF ELISA Kit, Mif ELISA Kit, PHATRDRAFT_49660 ELISA Kit, LOC100136498 ELISA Kit, cl405_1 ELISA Kit, LOC100284546 ELISA Kit

Background: MIF (or macrophage migration inhibitory factor) was the first lymphokine/cytokine to be recognized in the pregenomics era.Regardless, it is one of the least understood of all inflammatory mediators. Mouse MIF is a 12.5 kDa, 115 amino acid (aa) nonglycosylated polypeptide that is synthesized without a signal sequence.Secretion occurs nonclassically via an ABCA1 transporter. The initiating Met is removed, leaving Pro as the first amino acid. The molecule consists of two alpha -helices and six beta -strands, four of which form a beta -sheet. The two remaining beta -strands interact with other MIF molecules, creating a trimer. Structure-function studies suggests MIF is bifunctional with segregated topology. The N- and C-termini mediate enzyme activity (in theory). Phenylpyruvate tautomerase activity (enol-to-keto) has been demonstrated and is dependent upon Pro at position Amino acids 3-23 have also been shown to be reminescent of a GST glutathione-binding domain. MIF has proinflammatory cytokine activity centered on aa ’ s 49 - 65. On fibroblasts, MIF induces, IL-1, IL-8 and MMP expression, on macrophages, MIFstimulates, NO production and TNF- alpha release folllowing IFN- gamma activation. Mouse MIF apparently acts through CD74 and CD44,likely in some form of trimeric interaction. Mouse MIF is active on human cells, while human MIF is active on mouse cells. Mouse MIF is 99%, 84%, 90%, and 90% aa identical to rat, porcine, bovine and human MIF, respectively.

Pathways: Regulation of Systemic Arterial Blood Pressure by Hormones, Positive Regulation of Immune Effector Process, Production of Molecular Mediator of Immune Response, Regulation of Carbohydrate Metabolic Process, Feeding Behaviour, Smooth Muscle Cell Migration, Negative Regulation of intrinsic apoptotic Signaling