Lipocalin 2 ELISA Kit

Catalog No. ABIN455995

Product Details Lipocalin 2 ELISA Kit

Target: Lipocalin 2 (LCN2)

Reactivity: Human

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 in vitro quantitative determination of human neutrophil gelatinase-associated lipocalin,NGAL concentrations in serum, plasma and other biological fluids.

Sample Type: Plasma, Serum

Analytical Method: Quantitative

Specificity: This assay recognizes recombinant and natural human NGAL.

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

Characteristics: Homo sapiens,Human,Neutrophil gelatinase-associated lipocalin,NGAL,25 kDa alpha-2-microglobulin-related subunit of MMP-9,Lipocalin-2,Oncogene 24p3,p25,LCN2,HNL,NGAL,p25

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 NGAL. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for NGAL and 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 NGAL, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of NGAL 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 (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 × g. Remove serum and assay immediately or aliquot and store samples at -20 C or -80 C . Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 × g at 2 - 8 C within 30 minutes of collection. Store samples at -20 C or -80 C . Avoid repeated freeze-thaw cycles. Other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20 C or -80 C . Avoid repeated freeze-thaw cycles. Note: Serum and plasma to be used within 7 days may be stored at 2-8 C , otherwise samples must stored at -20 C ( ≤ 1 months) or -80 C ( ≤ 2 months) to avoid loss of 3 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.
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.

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 Lipocalin 2 ELISA Kit (ABIN455995)

Wu, Wang, Lv, Qin, Lei, Yuan, Wang: "The changes of serum sKlotho and NGAL levels and their correlation in type 2 diabetes mellitus patients with different stages of urinary albumin." in: Diabetes research and clinical practice, Vol. 106, Issue 2, pp. 343-50, (2014) (PubMed).

Target Details for Lipocalin 2

Target: Lipocalin 2 (LCN2)

Alternative Name: LCN2 (LCN2 Products)

Synonyms: LCN2 ELISA Kit, 24p3 ELISA Kit, MSFI ELISA Kit, NGAL ELISA Kit, AW212229 ELISA Kit, Sip24 ELISA Kit, lipocalin 2 ELISA Kit, LCN2 ELISA Kit, Lcn2 ELISA Kit

Background: NGAL (neutrophil gelatinase-associated lipocalin) belongs to the lipocalin family of proteins. These are typically small secreted proteins characterized by their ability to bind small, hydrophobic molecules in a structurally conserved pocket formed by beta -pleated sheet, to bind to specific cell-surface receptors and to form macromolecular complexes. NGAL has many synonyms: it also known as NL (neutrophil lipocalin, HNL: rat NL), lipocalin, oncogene protein 24p3 or uterocalin (in the mouse) and neu-related lipocalin or 25 kDa alpha 2-microglobulin-related protein6 (in the rat). Rat NGAL consists of a single disulfide-bridged polypeptide chain of 178 amino-acid residues with a calculated molecular mass of 22 kDa, but glycosylation increases its apparent molecular mass to 25 kDa. In neutrophils (neutrophilic polymorphonuclear leukocytes) and urine it occurs as monomer, with a small percentage of dimer and trimer, and also in complex with 92-kDa rat neutrophil type IV collagenase, also called gelatinase B or matrix metalloproteinase-9 (MMP-9). NGAL was originally isolated from the supernatant of activated rat neutrophils, but it is also expressed at a low level in other rat tissues including the kidney, prostate and epithelia of the respiratory and alimentary tracts. It is strongly expressed in adenomas and inflamed epithelia of the bowel, adenocarcinomas of the breast, and urothelial carcinomas. Because of its small molecular size and resistance to degradation, NGAL is readily excreted and detected in the urine, both in its free form and in complex with MMP-9. Urinary levels correlate with plasma or serum levels whatever the cause of increased NGAL production (AntibodyShop data), but particularly high urinary levels can be expected when it is released directly into the urine by the kidney tubules or urothelial carcinomas. It is uncertain how far NGALMMP-9 complexes from sources remote from the urinary tract are excreted as such into the urine or reform in the urine after independent excretion of NGAL and MMP-9. 2.

Pathways: Cellular Response to Molecule of Bacterial Origin, Transition Metal Ion Homeostasis