1

Human Fibroblast growth factor 23 ELISA
MATERIAL REQUIRED BUT NOT SUPPLIED

Cat. No.: BA1019
Enzyme Immunoassay for the quantitative determination of Fibroblast
growth factor 23 (FGF23) in human EDTA plasma.
Fibroblast growth factor-23 (FGF-23) is a phosphaturic hormone involved inmineral bone metabolism that helps control phosphate homeostasis andreduces 1,25- dihydroxyvitamin D synthesis. Recent data have highlighted
the relevant direct FGF-23 effects on the myocardium, and high plasma
levels of FGF-23 have been associated with adverse cardiovascularoutcomes in humans, such as heart failure and arrhythmias. Therefore, FGF23 has emerged as a novel biomarker of cardiovascular risk. experimental
data suggest FGF-23 as a direct mediator of cardiac hypertrophy
development, cardiac fibrosis and cardiac dysfunction via specificmyocardial FGF receptor (FGFR) activation.1

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5.

WARNINGS AND PRECAUTIONS
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2.
3.
4.
5.

PRINCIPLE OF FGF23 ELISA
The microtiter plate is coated with the antibody specifically binding
Fibroblast growth factor 23. The human EDTA plasma is incubated in theplate with the capture antibody.
The specimen is washed out and the specifically bound protein is incubatedwith biotin-labelled detection antibody. Following another washing step,
Streptavidin-HRP conjugate is added into the well.
Unbound reagent is then washed out. Horseradish peroxidase (HRP) boundin the complex reacts with the chromogenic substrate (TMB) creating theblue colour. The reaction is stopped by addition of STOP solution (H2SO4).
The absorbance values are measured at 450 nm (optionally 450/630 nm)
and are proportional to the concentration of FGF23 in the specimen. The
concentration of FGF23 in unknown samples is determined from thecalibration curve which is created by plotting the absorbance values against
the standard concentration values.

Glassware and test tubes
Microtiter plate washer
Precision pipettes (various volumes) with tips
Orbital shaker
Microtiter plate reader capable of measuring absorbanceat 450 nm or 450/630 nm with software for data generation

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7.

For research use only
For professional laboratory use
The reagents with different lot numbers should not be mixed
To prevent cross sample contamination, use disposable labware
and pipette tips
To protect laboratory stuff, wear protective gloves andprotective clothing
The substrate solution should remain colourless, keep it
protected from light
The test should be performed at standard laboratory conditions(temperature 25oC ± 2oC).

STORAGE CONDITIONS
1.
2.

The kit must be stored at 2 – 8oC.
The opened components can be stored for one week at 2 – 8oC.

PREPARATION OF REAGENTS

Use new pipette tip for pipetting different reagents and samples
to prevent cross-contamination.
All reagents and samples should be allowed to reach thetemperature 25oC ± 2oC.

Preparation of Standards
Reconstitute lyophilized Human FGF23 Standard in Dilution Buffer, for thevolume information see the Certificate of Analysis. Let it rehydrate for
15 min. The concentration of human FGF23 in Master Standard is2000 pg/mL.
Prepare set of Standard solution as follows:
Use the Master Standard for serial dilution (as below). Mix each tube
thoroughly before the next transfer. The Dilution Buffer serves as Blank.
Volume of Standard

Kit Contents
Item
Antibody Coated Microtiter Plate
Biotin-labelled Antibody
Streptavidin-HRP Conjugate
Master Standard (lyophilized)
Quality Control A (human serum, lyophilized)
Quality Control B (human serum, lyophilized)
Dilution Buffer
Wash Buffer 15x conc.
Substrate Solution
STOP Solution
v_01

Qty.
96 wells
13 mL
13 mL
1 vial1 vial1 vial
13 mL
50 mL
13 mL
13 mL

Std1

Standard 2000 pg/mL (lyophilized)

Dilution
BufferSee CofA

Concentration

Std2300 µL of Std1300 µL

1000 pg/mL

Std3

180 µL of Std2

450 µL400 pg/mL

Std4300 µL of Std3

300 µL200 pg/mL

Std5300 µL of Std4300 µL

100 pg/mL

Std6300 µL of Std5300 µL50 pg/mLBlank-300 µL

0 pg/mL

2000 pg/mL

Preparation of Quality Control A and B
Reconstitute the lyophilized human serum Quality Controls withdeionized/distilled water, for the volume information see the Certificate ofAnalysis. Let the QCs rehydrate for 15 min and dilute them 1:2 in Dilution
Buffer, prior to use, see Preparation of samples.

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Preparation of Wash Buffer 1x3.

Prepare a working solution of Wash Buffer by adding 50 mL of Wash Buffer
15x conc. to 700 mL of deionized/ distilled water (dH2O). Mix well. Store at4oC for two weeks or at -20oC for long term storage.

Accuracy

Dilution linearity
SamplePreparation of samples

Dilution

Measuredconcentration(pg/mL)
2003
1058
544
284
441
231
111
61

Expected
concentration(pg/mL)
1001
501
250
221
110
55

Yield(%)

Measuredconcentration(pg/mL)
61
749
421
225

Expected
concentration(pg/mL)
861
461
261

Yield(%)

1Human EDTA plasma may be used with this assay. For long-term storage thesamples should be frozen at minimum -70oC. Lipemic or haemolytic samples
may cause false results.

2x4x8x2

Recommended dilution of samples is 1:2, i.e., for singlets 75 µL of sample +75 µL of Dilution Buffer, for duplicates 150 µL of samples + 150 µL of Dilution
Buffer, respectively.

2x4x8x106
109
113
105101110

Do not store the diluted samples.
Spiking Recovery

ASSAY PROCEDURE
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Prepare the reagents as described in the previous chapter.
Pipette 100 µL of set of Standards, Quality Controls, diluted
Samples and Dilution Buffer = Blank into each well. Incubate forOVER NIGHT at 4oC ±2oC, NO shaking.
3. Wash the wells 5-times with 1x Wash Buffer (350 µL/well). When
finished, tap the plate against the paper towel to remove theliquid completely.
4. Pipette 100 µL of Biotin-labelled Antibody into each well.
Incubate for 2 hours at 25oC ±2oC, shaking at 300 rpm.
5. Wash the wells as described in point 3.
6. Pipette 100 µL of Streptavidin-HRP into each well. Incubate for
30 min at 25oC ±2oC, shaking at 300 rpm.
7. Wash the wells as described in point 3.
8. Pipette 100 µL Substrate solution, incubate for 15 min,at 25oC ±2oC. Avoid exposure to the light during this step.
9. Pipette 100 µL of STOP solution.
10. Read the signal at 450 or 450/630 nm within 15 min.

SampleSpike(pg/mL)

1800
400200

Typical standard curve
The standard curve needs to be measured in every test. Most of themicroplate reader can automatically calculate the analyte concentration
using 4-parameter algorithm or alternative functions to fit the standard
points properly. The concentrations need to be multiplied by the dilution
factor, either automatically by reader or manually.

Human FGF23 Standard Curve
4Plate layout
12Std 1
Std 2
Std 3
Std 4
Std 5
Std 6
QCA
QCB3Bckg

4
Sa 45
Sa 8

Sa 1

Sa 5

Sa 9

Sa 2

Sa 6

Sa3

Sa 7

Sa10
Sa116
Sa12
Sa
13
Sa
14
Sa
157
Sa
16
Sa
17
Sa
18
Sa
198
Sa
20
Sa
21
Sa
22
Sa
23

9
Sa
24
Sa
25
Sa
26
Sa
27

10
Sa
28
Sa
29
Sa
30
Sa
31

11
Sa
32
Sa
33
Sa
34
Sa
35

12
Sa
36
Sa37
Sa38
Sa
39

PERFORMANCE CHARACTERISTICS
Samples used in the tests were diluted 1:2 as recommended and assayed.
The results are multiplied by the dilution factor.
1.

Sensitivity

3.5
3Absorbance (450/630 nm)

AB
CDEFGH

2.5
2
1.5
10.5
050

The limit of detection, defined as a concentration of human FGF23 giving
absorbance higher than absorbance of blank + 3 standard deviations, isbetter than 12.5 pg/mL of sample.
2.

v_01

200

400

1000

2000

Concentration (pg/mL)

1 Vázquez-Sánchez S, Poveda J, Navarro-García JA, González-Lafuente L, Rodríguez-Mean (pg/mL)
1718
492

SD
100
19CV (%)
5.8
3.8SD
390

CV (%)
6.3
12.1Inter-assay (Run – to – run)
Sample12100

RESOURCES

Precision

Intra-assay
Sample12879186

Mean (pg/mL)
41742

Sánchez E, Ruilope LM, Ruiz-Hurtado G. An Overview of FGF-23 as a Novel Candidate
Biomarker of Cardiovascular Risk. Front Physiol. 2021 Mar 9;12:632260. doi:10.3389/fphys.2021.632260. PMID: 33767635; PMCID: PMC7985069.