ImmunoDiagnostics Mouse Lipocalin-2 ELISA kit

Reagents Supplied

Each kit is sufficient for one 96-well plate and contains the following components:

1. Microtiter strips (96 wells) – Coated with anti-mouse LCN2 polyclonal antibody, sealed.
2. 10×Wash buffer, 50ml.
3. 5×Assay buffer, 30ml.
4. 100×Detection antibody solution – A biotin labelled polyclonal antibody against mouse LCN2, 0.12ml.
5. 200×STP-HRP solution, 0.06ml.
6. Mouse LCN2 standard, 10ng of recombinant mouse LCN2 in a buffered protein base, lyophilized.
7. Substrate solution, 12ml, ready for use.
8. Stop solution, 12ml, ready for use.

Other Materials Required, But Not Provided

1. Pipettes and pipette tips.
2. 96-well plate or manual strip washer.
3. Buffer and reagent reservoirs.
4. Paper towels or absorbent paper.
5. Plate reader capable of reading absorbency at 450nm.
6. Distilled water or deionized water.

Storage

The kit should be stored at 2-8°C upon receipt, and all reagents should be equilibrated to room temperature before use. Remove any unused antibody-coated strips from the mouse LCN2 microplate, return them to the foil pouch and reseal. Once opened, the strips may be stored at 2-8°C for up to one month.

Preparation of Reagents

Bring all reagents and materials to room temperature before assay.

1. 1×Assay buffer
   Prepare 1×Assay buffer by mixing the 5×Assay buffer (30ml) with 120ml of distilled water or deionized water. If precipitates are observed in the 5×Assay buffer bottle, warm the bottle in a 37°C water bath until the precipitates disappear. The 1×Assay buffer may be stored at 2-8°C for up to one month.
2. 1×Wash buffer
   Prepare 1×Wash buffer by mixing the 10×Wash buffer (50ml) with 450ml of distilled water or deionized water. If precipitates are observed in the 10×Wash buffer bottle, warm the bottle in a 37°C water bath until the precipitates disappear. The 1×Wash buffer may be stored at 2-8°C for up to one month.
3. 1×Detection antibody solution
   Spin down the 100×Detection antibody solution briefly and dilute the desired amount of the antibody 1:100 with 1×Assay buffer, 100µl of the 1×Detection antibody solution is required per well. Prepare only as much 1×Detection antibody solution as needed. Return the 100×Detection antibody solution to 2-8°C immediately after the necessary volume is removed.
4. 1×STP-HRP solution
   Spin down the 200×STP-HRP solution briefly and dilute the desired amount of the 200×STP-HRP solution 1:200 with 1×Assay buffer, 100µl of the 1×STP-HRP solution is required per well. Prepare only as much 1×STP-HRP solution as needed. Return the 200×STP-HRP solution to 2-8°C immediately after the necessary volume is removed.

Preparation of Standards and Samples

Mouse LCN2 standards: Reconstitute the lyophilized standard with 1ml distilled or deionized water. Allow at least 10 minutes for complete reconstitution and invert the vial several times to mix and vortex. This reconstitution produces a stock solution of 10ng/ml. Prepare serially diluted standards using 1×Assay buffer as follows:

1×Assay buffer serves as the zero standard (0ng/ml). The reconstituted standard stock should be aliquoted and frozen at -20ºC for one month. Avoid repeating freezing/thawing cycles. Please do not store the diluted standard solutions. Sample preparation Serum or plasma sample is generally required an 80-fold dilution in this assay. A suggested dilution step is to add 10µl of sample to 790µl of 1×Assay buffer. Cellular extract and culture media dilutions will vary and need to be optimized by the user, also use 1×Assay buffer to prepare these samples.

Assay Characteristics

Sensitivity

The lowest level of mouse LCN2 that can be detected by this assay is 0.156ng/ml.

Specificity

The antibodies used in this assay are specific to mouse LCN2 and do not cross-react with human LCN2, and other cytokine or hormone molecules.

Assay Principle

This assay is a sandwich ELISA using antigen-affinity-purified polyclonal antibodies against mouse LCN2. The immunoplate is precoated with anti-mouse LCN2 capture antibody. Standards and samples are added to the wells and any mouse LCN2 present is captured by the immobilized antibody. After wash step to remove any unbound substances, a biotin labelled anti-mouse LCN2 detection antibody is added. After washing procedure, streptavidinHRP conjugate (STP-HRP) is added. After the last wash step, an HRP substrate solution is added and color develops in proportion to the amount of mouse LCN2 bound initially. The assay is stopped and the optical density of the wells determined using a microplate reader. Since the increases in absorbance are directly proportional to the amount of captured mouse LCN2, the unknown sample concentration can be interpolated from a reference curve included in each assay.

Assay Procedure

It is recommended that all standards and samples be assayed in duplicate.

1. Add 100µl of standard or sample per well, incubate at room temperature for 1 hour.
2. Discard the content and tap the plate on a clean paper towel to remove residual solution in each well. Add 300µl of 1×Wash buffer to each well and incubate for 1 minute. Discard the 1×Wash buffer and tap the plate on a clean paper towel to remove residual wash buffer. Repeat the wash step for a total 3 washes.
3. Add 100µl of 1×Detection antibody solution to each well, incubate at room temperature for 1 hour.
4. Wash each well 3 times as in step 2.
5. Add 100µl of 1×STP-HRP solution to each well and incubate at room temperature for 20 minutes.
6. Wash each well 4 times as described in step 2.
7. Add 100µl of Substrate solution to each well, incubate at room temperature for 15 minutes. Protect from light.
8. Add 100µl of Stop solution to each well, gently tap the plate frame for a few seconds to ensure thorough mixing.
9. Measure absorbance of each well at 450nm immediately.

Calculation

1. Subtract the absorbance of the blank from that of standards and samples.
2. Generate a standard curve by plotting the absorbance obtained (y-axis) against mouse LCN2 concentrations (x-axis). The best fit line can be generated with any curve-fitting software by regression analysis. Any curve of 4-parameter or log-log curve fitting can be used for calculation.
3. Determine mouse LCN2 concentration of samples from standard curve and multiply the value by the dilution factor.

Assay Procedure Summary

Add 100µl of Standard or sample to each well.
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Incubate at room temperature for 1 hour.
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Aspirate and wash each well three times.
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Add 100µl of 1×Detection antibody solution to each well.
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Incubate at room temperature for 1 hour.
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Aspirate and wash each well three times.
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Add 100µl of 1×STP-HRP solution to each well.
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Incubate at room temperature for 20 minutes.
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Aspirate and wash each well four times.
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Add 100µl of Substrate solution to each well.
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Incubate at room temperature for 15 minutes.
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Add 100µl of Stop solution to each well.
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Measure absorbance of each well at 450nm.
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Calculation

The following standard curve is provided for demonstration only. A standard curve should be generated for each set of sample assay.

Mouse LCN2 standard curve (4-parameter)

Lipocalin-2, also known as neutrophil gelatinase-associated lipocalin (NGAL), 24p3, siderocalin, or neutrophil lipocalin (NL), is a secretory glycoprotein expressed in liver, lung, kidney, adipocytes, activated neutrophils and macrophages. Lipocalin-2 is upregulated under various inflammation and infection conditions and can be served as a sensitive biomarker for various renal injuries. Serum levels of lipocalin-2 are positively correlated with obesity, insulin resistance, hyperglycemia, coronary heart disease and fatty liver disease in humans.

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