Lipocalin 2 ELISA Kit

Catalog No. ABIN2014344

This Human Lipocalin 2 ELISA Kit is a Colorimetric ELISA Kit designed to quantify Human Lipocalin 2.

Quick Overview for Lipocalin 2 ELISA Kit (ABIN2014344)

Target: Lipocalin 2 (LCN2)

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 0.04-27 ng/mL

Application: ELISA

Sample Type: Plasma

Product Details Lipocalin 2 ELISA Kit

Minimum Detection Limit: 0.04 ng/mL

Purpose: This test kit is intended for use in the quantitative determination of human neutrophil gelatinase-associated lipocalin (Lipocalin-2 or NGAL) in EDTA-plasma. Indications for use: Patient may have a higher than normal level of NGAL with1. Acute kidney failure2. Systemic vasculitis3. Acute ischemic heart disease4. Other inflammatory diseases or infectious diseases

Analytical Method: Quantitative

Components: 1. Anti-NGAL Antibody Coated Microplate
One bottle contains 30 mL of 2-fold concentrated buffer matrix with protein stabilizers and preservative. This reagent should be stored at 2-8 °C and is stable until the expiration date on the kit box. Before use the concentrated buffer must be diluted with 30 mL of demineralized water and mixed well.

Material not included: 1. Precision single channel pipettes capable of delivering 100 µL.
2. Disposable pipette tips suitable for above volume dispensing.
3. Aluminum foil.
4. Deionized or distilled water.
5. Plastic microtiter well cover or polyethylene film.
6. ELISA multichannel wash bottle or automatic (semi-automatic) washing system.
7. Spectrophotometric microplate reader capable of reading absorbance at 450/650 or 450/620 nm.

Application Details

Sample Volume: 10 μL

Assay Time: 4 h

Plate: Pre-coated

Protocol: This ELISA kit is designed, developed and produced for the quantitative measurement of human NGAL in EDTA-plasma samples. The assay utilizes the sandwich technique with selected antibodies that bind to various epitopes of NGAL.Assay standards, controls and patient samples are added directly to wells of a microtiter plate that is coated with antibody to human NGAL and incubated at room temperature for one hour. The plate is then washed and horseradish peroxidase (HRP) conjugated anti NGAL is added to each well. After an additional incubation period, a sandwich of solid-phase polyclonal antibody - human NGAL HRP-conjugated antibody is formed. The unbound antibodies and buffer matrix are removed in the subsequent washing step. For the detection of this immunocomplex, the well is then incubated with a substrate solution in a timed reaction, which is terminated with an acidic reagent (i.e. ELISA stop solution). The absorbance is then measured in a spectrophotometric microplate reader. The enzymatic activity of the immunocomplex bound to the wall of each microtiter well is directly proportional to the amount of human NGAL in the test sample. A standard curve is generated by plotting the absorbance versus the respective human NGAL concentration for each standard on a point-to-point or 4-parameter curve fitting. The concentration of human NGAL in test samples is determined directly from this standard curve.

Reagent Preparation:

(1) Prior to use allow all reagents to come to room temperature. Reagents from different kit lot numbers should not be combined or interchanged.
(2) ELISA Wash Concentrate must be diluted to working solution prior to use. Please see REAGENTS section for details.
(3) Reconstitute assay standards and controls by adding 1.0 mL of deminerialized water to each standard and control bottle. Allow the standard and controls to sit undisturbed for 5 minutes, and then mix well by inversions or gentle vortexing. Make sure that all solid is dissolved completely prior to use. These reconstituted standards and controls may be stored at 2- 8 C for up to 3 days or below ?20 C for long-term storage. Do not exceed 3 freeze-thaw cycles.
(4) Concentrated Patient Sample Diluent must be diluted to working solution prior to use. Please see REAGENTS section for details.
(5) Each unknown sample needs to be diluted 1:100 using 1x NGAL Sample Dilution Buffer as a sample diluent.
(6) Prepare Tracer Antibody working solution by 1:21 fold dilution of the NGAL Tracer Antibody by adding the tracer antibody into the Tracer Antibody Diluent . Following is a table that outlines the relationship of strips used and antibody mixture prepared. NOTE: the tracer antibody should be prepared just prior to the end of the first incubation cycle.

Sample Collection: EDTA-plasma samples are suitable specimens for human NGAL measurement. Only 10 µL of human EDTA-plasma is required for a duplicate determination of human NGAL with this test kit. No special preparation of individual is necessary prior to specimen collection. EDTA-plasma should be collected by standard technologies of clinical laboratory practice and recommended by manufacturer of sample collection tube. It is extremely important to carefully separate the plasma from blood cells to avoid hemolyzation, etc. EDTA-plasma should be transferred to a clean test tube right after centrifugation. EDTA-plasma samples should be stored at 2 ? 8 C if the assay is to be performed within 72 hours. Otherwise, patient samples should be stored at -20 °C or below until measurement. Avoid more than three times freeze-thaw cycles of specimen. Do not use hemolyzed, hyperlipermic, heat-treated or any contaminated specimens. Serum sample should not be used for NGAL measurement because the blood clotting process may lead to release NGAL from neutriphils, which could result in an unreliable test results. Samples of heparin plasma and citrate plasma may be used for NGAL measurement.

Assay Procedure:

(1) Add 100 µL of Standards, Controls and diluted patient samples (diluted beforehand 1:100 with NGAL Sample Dilution Buffer, Cat. 30654) into the designated microwells.
(2) Seal the plate wells securely, cover with foil or other material to protect from light. Incubate the plate static, at room temperature for 1 hr. 5 minutes.
(3) Wash each well 5 times by dispensing 350 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
(4) Dilute the proper amount of Tracer Antibody for the assay.
(5) Add 100 µL of the above Tracer Antibody to each well.
(6) Seal the plate wells securely, cover with foil or other material to protect from light. Incubate the plate static, at room temperature for 30 minutes 5 minutes.
(7) Wash each well 5 times by dispensing 350 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
(8) Add 100 µL of ELISA HRP Substrate into each of the wells.
(9) Cover the plate with aluminum foil or other material to avoid exposure to light. Incubate the plate static, at room temperature for 20 minutes.
(10) Immediately add 100 µL of ELISA Stop Solution into each of the wells. Mix gently.
(11) Read the absorbance at 450 nm with reference filter at 620 nm or 650 nm.

Calculation of Results:

It is recommended to use a point-to-point or 4-parameter standard curve fitting.
1. Calculate the average absorbance for each pair of duplicate test results.
2. Subtract the average absorbance of the level 1 standard (0 ng/mL) from the average absorbance of all other readings to obtain corrected absorbance.
3. The standard curve is generated by the corrected absorbance of all standard levels on the ordinate against the standard concentration on the abscissa using point-to-point or log-log paper. Appropriate computer assisted data reduction programs may also be used for the calculation of results. The human NGAL concentrations for the controls and the patient samples are read directly from the standard curve using their respective corrected absorbance.

Assay Precision: The intra-assay precision was validated by measuring three diluted 1:100 control samples with 16 replicate determinations. The inter-assay precision was validated by measuring two control levels in duplicate in 14 individual assays.

Restrictions: For Research Use only

Handling

Precaution of Use: The reagents must be used in professional laboratory. Source material for reagents containing bovine serum was derived in the contiguous 48 United States. It was obtained only from healthy donor animals maintained under veterinary supervision and found free of contagious diseases. Wear gloves while performing this assay and handle these reagents as if they are potentially infectious. Avoid contact with reagents containing TMB, hydrogen peroxide, or sulfuric acid. TMB may cause irritation to skin and mucous membranes and cause an allergic skin reaction. TMB is a suspected carcinogen. Sulfuric acid may cause severe irritation on contact with skin. Do not get in eyes, on skin, or on clothing. Do not ingest or inhale fumes. On contact, flush with copious amounts of water for at least 15 minutes. Use Good Laboratory Practices.

Storage: 4 °C

Target Details for Lipocalin 2

Target: Lipocalin 2 (LCN2)

Alternative Name: Neutrophil Gelatinase-Associated Lipocalin

Background: NGAL or neutrophil gelatinase-associated lipocalin also known as Lipocalin-2 (LCN2) or oncogene 24p3 is a protein, which in humans is encoded by the LCN2 gene. NGAL is involved in innate immunity by sequestrating iron that in turn limits bacterial growth. It is expressed in neutrophils and in low levels in the kidney, prostate, and epithelia of the respiratory and alimentary tracts. Studies have shown that NGAL is an early biomarker for ischaemic renal injury after cardiopulmonary bypass.

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