ImmunoDiagnostics Mouse FGF-21 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 a polyclonal antibody against mouse FGF-21, sealed.
2. 10×Wash buffer, 50ml.
3. 5×Assay buffer, 20ml.
4. 100×Detection antibody solution – A biotin labelled polyclonal antibody against mouse FGF-21, 0.12ml.
5. Mouse FGF-21 standard, 2000pg of recombinant Mouse FGF-21in a buffered protein base, lyophilized.
6. 200×STP-HRP solution, 0.06ml.
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 antibodycoated strips from the mouse FGF-21 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 (20ml) with 80ml 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 FGF-21 standards: Reconstitute the lyophilized standard with 1 ml of 1×Assay buffer to generate a standard stock solution of 2000pg/ml. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare serially diluted standards using 1×Assay buffer as follows:

1x Assay buffer serves as the zero standard (0ng/ml).

Note: The reconstituted standard stock should be aliquoted and frozen at -80ºC 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 a 2-fold dilution in 1×Assay buffer.

Assay Characteristics

Assay Range

15.6-2000pg/ml

Sensitivity

The lowest level of mouse FGF-21 that can be detected by this assay is 15.6pg/ml.

Specificity

Cross reactivity of recombinant proteins

Precision

Intra-assay Precision (Precision within an assay) C.V <8%.

Inter-assay Precision (Precision between assays) C.V <10%.

Recovery

The recovery of the assay was determined by adding various amounts FGF21 to a sample. The measured concentration of the spiked sample in the assay was compared to the expected concentration. The average recovery was 92%.

Linearity

To assess the linearity of the assay, a sample with high level of FGF-21 was serially diluted with the 1×Assay buffer to produce samples with values within the dynamic range of the assay.

Assay Principle

This assay is a quantitative sandwich ELISA. The immunoplate is precoated with a rabbit polyclonal antibody specific for mouse FGF-21. Standards and samples are pipetted into the wells and any mouse FGF-21 present is bound by the immobilized antibody. After washing away any unbound substances, a biotin labelled polyclonal antibody specific for mouse FGF-21 is added to the wells. After wash step to remove any unbound reagents, streptavidin-HRP 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 FGF-21 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 FGF-21, 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, 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 FGF-21 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 FGF-21 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 FGF-21 standard curve (4-parameter)

Fibroblast growth factor 21(FGF-21) is a novel protein that has been implicated in the regulation of lipid and glucose metabolism under fasting and ketotic conditions. In murine models, FGF-21 is predominantly expressed in liver, but it also expressed in adipose tissue and pancreatic βcells. FGF-21 stimulates glucose uptake in adipocytes. It also protects animals from diet-induced obesity when overexpressed in transgenic mice and lowers blood glucose and triglyceride levels when administered to diabetic rodents. When administered daily for 6 weeks to diabetic rhesus monkeys, FGF-21 caused a dramatic decline in fasting plasma glucose, fructosamine, triglycerides, insulin, and glucagon. Furthermore, elevated plasma FGF-21 concentrations in humans appear to be related to the presence of hepatic and peripheral insulin resistance

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