Developed & Manufactured by Biomedica

FGF23 (C-terminal) multi-matrix ELISA

Cat. No.: BI-20702

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Therapeutic Areas
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Assay Characteristics
  • Category number

    BI-20702

  • Method

    Sandwich ELISA, HRP/TMB, 12×8-well detachable strips

  • Sample type

    Serum, EDTA plasma, citrate plasma, heparin plasma

  • Sample volume

    50 µl / well

  • Assay time

    20-24 h / 1 h / 30 min

  • Sensitivity

    0.08 pmol/l (= 0.6 pg/ml)

  • Standard range

    0 – 20 pmol/l (= 0 – 154 pg/ml)

  • Conversion factor

    1 pg/ml = 0.133 pmol/l (MW: 7.52 kDa)

  • Specificity

    Human FGF23 (Intact and C-terminal fragments of endogenous and recombinant human FGF23)

  • Precision

    In-between (n=10): ≤ 10 % CV

    Within-run (n=6): ≤ 12 % CV

  • Use

    CE marked – for IVD use in the EU

  • Validation Data

    See validation data tab for: precision, accuracy, dilution linearity, values for healthy donors, etc

Product details

Product Overview

The FGF23 (C-terminal) immunoassay is an overnight, 96-well sandwich ELISA for the quantitative determination of FGF23 in human serum and plasma. The assay employs human serum-based standards to ensure the measurement of biologically reliable data.

The FGF23 (C-terminal) kit uses antibodies with characterized binding affinities.

Principle Of The Assay

The FGF23 (C-terminal) ELISA kit is a sandwich enzyme immunoassay for the quantitative determination of FGF23 in human serum and plasma samples.

Figure explaining the principle of a sandwich ELISA:



In a first step, standard/control/sample and detection antibody (biotinylated polyclonal rabbit anti-human FGF23) are pipetted into the wells of the microtiter strips, which are pre-coated with anti-human FGF23 antibody. FGF23 present in the standard/control/sample binds to the pre-coated antibody in the well and forms a sandwich with the detection antibody. In the washing step all non-specific unbound material is removed. In a second step, the conjugate (streptavidin-HRP) is pipetted into the wells and reacts with the detection antibody. After another washing step, the substrate (TMB, tetramethylbenzidine) is pipetted into the wells. The enzyme-catalyzed color change of the substrate is directly proportional to the amount of FGF23. This color change is detectable with a standard microtiter plate reader. A dose response curve of the absorbance (optical density, OD at 450 nm) vs. standard concentration is generated, using the values obtained from the standard. The concentration of FGF23 in the sample is determined directly from the dose-response curve.

Typical Standard Curve

The figure below shows a typical standard curve for the FGF23 (C-terminal) ELISA. The immunoassay is calibrated against recombinant C-terminal FGF23 peptide:



ELISA Kit Components

Contents

Description

Quantity

PLATE

Goat polyclonal anti-FGF23 antibody pre-coated microtiter strips in strip holder, packed in aluminum bag with desiccant

12 x 8 tests

WASHBUF

Wash buffer concentrate 20x, natural cap

1 x 50 ml

STD

Standards 1-7, (0; 0.2; 0.6; 1.8; 5; 10; 20 pmol/l), recombinant human C-terminal FGF23 peptide in human serum, white caps, lyophilized

7 vials

CTRL

Control A + B, yellow caps, lyophilized, exact concentrations see labels

2 vials

AB

Rabbit polyclonal anti-FGF23 antibody – biotin labeled, green cap, green dye, ready to use

1 x 6 ml

ASYBUF

Assay buffer, red cap, ready to use

1 x 20 ml

CONJ

Conjugate (streptavidin-HRP), amber bottle, amber cap, ready to use

1 x 13 ml

SUB

Substrate (TMB solution), amber bottle, blue cap, ready to use

1 x 13 ml

STOP

Stop solution, white cap, ready to use

1 x 7 ml

Storage instructions: All reagents of the FGF23 (C-terminal) ELISA kit are stable at 2-8°C until the expiry date stated on the label of each reagent.

Instructions for use

Sample Collection & Storage

Serum, EDTA plasma, heparin plasma and citrate plasma are suitable for use in this assay. Do not change sample type during studies. We recommend duplicate measurements for all samples, standards and controls. The sample collection and storage conditions listed are intended as general guidelines.

Serum & Plasma

Collect venous blood samples in standardized serum separator tubes (SST) or standardized blood collection tubes using EDTA, heparin or citrate as an anticoagulant. For serum samples, allow samples to clot for 30 minutes at room temperature. Perform separation by centrifugation according to the tube manufacturer’s instructions for use. Assay the acquired samples immediately or aliquot and store at -25°C or lower. Lipemic or haemolyzed samples may give erroneous results. Samples are stable for at least four freeze-thaw cycles.

Reagent Preparation

Wash Buffer

1.

Bring the WASHBUF concentrate to room temperature. Crystals in the buffer concentrate will dissolve at room temperature.

2.

Dilute the WASHBUF concentrate 1:20, e.g. 50 ml WASHBUF + 950 ml distilled or deionized water. Only use diluted WASHBUF when performing the assay.

The diluted WASHBUF is stable up to one month at 4°C (2-8°C).

Standards & Controls For Serum and Plasma Measurements

1.

Pipette 400 µl of distilled or deionized water into each standard (STD) and control (CTRL) vial. The exact concentration is printed on the label of each vial.

2.

Leave at room temperature (18-26°C) for 15 min. Vortex gently.

Reconstituted STDs and CTRLs are stable for 4 h at room temperature (18-24°C) or at -25°C or lower until expiry date stated on the label. STDs and CTRLs are stable for three freeze-thaw cycles.

Sample Preparation

Bring samples to room temperature and mix gently to ensure the samples are homogenous. We recommend duplicate measurements for all samples.

Samples for which the OD value exceeds the highest point of the standard range can be diluted with ASYBUF (assay buffer). If dilution is required, we recommend to dilute serum samples 1:11 (1+10, e.g. 10 µl sample + 100 µl ASYBUF) and plasma samples 1:41 (1+40, e.g. 10 µl sample + 400 µl ASYBUF). The kit includes sufficient ASYBUF for a 1:41 dilution of 40 samples. Additional ASYBUF can be ordered (cat. no. BI-201702-ASYBUF).

Assay Protocol

Read the entire protocol before beginning the assay.

1.

Bring samples and reagents to room temperature (18-24°C).

2.

 Mark positions for STD/CTRL/SAMPLE (standard/control/sample) on the protocol sheet.

3.

Take microtiter strips out of the aluminum bag. Store unused strips with desiccant at 4°C in the aluminum bag. Strips are stable until expiry date stated on the label.

4.

Pipette 50 µl STD/SAMPLE/CTRL (Standard/Sample/Control) in duplicate into respective well.

5.

Add 50 µl AB (biotinylated anti-FGF23 antibody, green cap, green dye) into each well, mix gently.

6.

Cover the plate tightly and incubate overnight (20-24 h) at room temperature (18-24°C).

7.

Aspirate and wash wells 5x with 300 µl diluted WASHBUF. After the final wash, remove remaining WASHBUF by strongly tapping the plate against a paper towel.

8.

Add 100 µl CONJ (conjugate, amber cap) into each well.

9.

Cover the plate tightly and incubate for 1 h at room temperature (18-24°C) in the dark.

10.

Aspirate and wash wells 5x with 300 µl diluted WASHBUF. After the final wash, remove remaining WASHBUF by strongly tapping the plate against a paper towel.

11.

Add 100 µl SUB (substrate, blue cap) into each well.

12.

Incubate for 30 min at room temperature (18-24°C) in the dark.

13.

Add 50 µl STOP (stop solution, white cap) into each well, mix gently.

14.

Measure absorbance immediately at 450 nm with reference 630 nm, if available.

Calculation of Results

Construct a standard curve from the absorbance read-outs of the standards using commercially available software capable of generating a four-parameter logistic (4-PL) fit. Alternatively, plot the standards’ concentration on the x-axis against the mean absorbance for each standard on the y-axis and draw a best fit curve through the points on the graph. Curve fitting algorithms other than 4-PL have not been validated and will need to be evaluated by the user.

Obtain sample concentrations from the standard curve. If required, pmol/l can be converted into pg/ml by applying a conversion factor (1 pg/ml = 0.133 pmol/l; MW: 7.52 kDa). Respective dilution factors have to be considered when calculating the final concentration of the sample.

The quality control protocol supplied with the kit shows the results of the final release QC for each kit lot. Data for optical density obtained by customers may differ due to various influences and/or due to the normal decrease of signal intensity during shelf life. However, this does not affect validity of results as long as an OD of 1.50 or more is obtained for the standard with the highest concentration and the values of the CTRLs are in range (target ranges see labels).

Background & Therapeutic Areas

INFORMATION ON THE ANALYTE

FGF23 protein

FGF23 (fibroblast growth factor 23) is a member of the fibroblast growth factor family and controls phosphate and vitamin D homeostasis. It is a 32 kDa protein of 251 amino acids including a 24 amino acid signal peptide. The N-terminal FGF homology region of FGF23 is separated from the unique C-terminal region by a proteolytic cleavage site. A proportion of FG23 is proteolytically processed between arginine179 and serine180 to generate N-terminal and C-terminal fragments. Therefore, the major forms of FGF23 present in human circulation are hormonally intact FGF23 and inactive N-terminal and C-terminal fragments. FGF23 binds to FGF receptor 1c (FGFR1c) with its N-terminal region, while the C-terminal region is capable of interacting with the co-receptor αKlotho to confer high-affinity binding to the receptor. FGFR1c and αKlotho are expressed in the distal nephron and the parathyroid gland. Co-receptor independent signaling of FGF23 has been described for other FGFRs, which are expressed in a variety of tissues. The main source of FGF23 are osteocytes in the bone.

Molecular Weight

7.52 kDa (C-terminal fragment of FGF23)

Cellular localization

Extracellular

Post-translational modifications

Glycosylation, phosphorylation

Sequence similarities

Member of the fibroblast growth factor (FGF) family

Alternative Names

Fibroblast growth factor 23, ADHR, FGFN, HPDR2, HYPF, PHPTC, HFTC2

Entrez/NCBI ID

8047 link: https://www.ncbi.nlm.nih.gov/gene/8074

Genecards

GC12M004347 link: https://www.genecards.org/cgi-bin/carddisp.pl?gene=FGF23

OMIM

605380 link: https://www.omim.org/entry/605380

PDB

2P39 link: https://www.rcsb.org/structure/2P39

Pfam

PF00167 link: https://pfam.xfam.org/family/PF00167

Protein Atlas

FGF23 link: https://www.proteinatlas.org/ENSG00000118972-FGF23/tissue

Uniprot ID

Q9GZV9 link: https://www.uniprot.org/uniprot/Q9GZV9

FGF23 Function

FGF23 is an endocrine hormone regulating phosphate homeostasis by modulating renal phosphate reabsorption, vitamin D metabolism and parathyroid hormone (PTH) secretion. Increased dietary phosphate intake or reduced phosphate excretion due to renal insufficiency stimulates FGF23 secretion in the bone. FGF23 induces the internalization of phosphate transporters in the kidney and thus, reduces phosphate reabsorption. The subsequent renal excretion of phosphate (phosphaturia) restores phosphate balance. PTH and active vitamin D (1,25-dihydroxyvitamin D) both stimulate FGF23 secretion and are suppressed by rising FGF23 levels in a negative feedback loop. FGF23 inhibits the activation of 25-hydroxyvitamin D into 1,25 dihydroxyvitamin D through downregulation of the enzyme 1α-hydroxylase catalyzing this metabolic step. At the same time, FGF23 promotes the upregulation of 24-hydroxylase, an enzyme mediating the inactivation of 1,25 dihydroxyvitamin D. FGF23-induced downregulation of active vitamin D limits the gastrointestinal uptake of phosphate and thus, contributes to the phosphate lowering effects of FGF23. Since PTH releases phosphate from bone, FGF23-mediated suppression of PTH secretion builds the third axis by which phosphate homeostasis is controlled by FGF23.

In humans, circulating levels of FGF23 can be altered in both genetic and acquired diseases. A primary excess in circulating intact FGF23 is the underlying cause of diseases like X-linked hypophosphatemic rickets (XLH), autosomal dominant/recessive hypophosphatemic rickets (ADHR/ARHR) or tumor-induced osteomalacia. The excessive FGF23 levels in these diseases cause renal phosphate wasting, low active vitamin D concentrations and defective mineralization of bones. In case of a genetic disease, the mutations accounting for hyperphosphatemia or hypophosphatemia are located in either the FGF23 gene itself or one of its regulators. In ADHR, missense mutations in the FGF23 gene inhibit the cleavage into N- and C-terminal fragments and thus, increase the amount of circulating intact FGF23. However, the exact molecular mechanisms by which some mutations other than in the FGF23 gene itself are causing FGF23-related disease is not yet fully understood.

Circulating FGF23 is also elevated in patients with chronic kidney disease (CKD), presumably in response to decreased renal excretion of phosphate. During the course of the disease, FGF23 secretion is upregulated and the ratio between C-terminal and intact FGF23 is shifted towards the intact protein. Simultaneous upregulation of FGF23 cleavage in osteocytes maintains near-normal levels of biologically active, intact circulating FGF23, whereas downregulated or impaired FGF23 cleavage may contribute to elevated intact serum FGF23 in CKD. The increase in FGF23 concentration in CKD patients is accompanied by low levels of active vitamin D and secondary hyperparathyroidism. Increased FGF23 in CKD patients is not only associated with disease progression, but also with left ventricular hypertrophy and endothelial dysfunction.

Besides hyperphophatemia, other conditions associated with CKD such as acidosis, tissue hypoxia, iron deficiency, and inflammation may also contribute to the increase in FGF23 levels. Iron stimulates FGF23 production and is counterbalanced by increased cleavage of newly synthesized FGF23 within healthy osteocytes. As a result, FGF23 fragments are elevated, but serum phosphate levels remain normal due to normal circulating levels of biologically active FGF23. Inflammation plays an essential role in FGF23 regulation as it triggers FGF23 expression and cleavage thus revealing its pro-inflammatory and immune-modulatory properties.

Epidemiological data suggests that higher FGF23 concentrations are associated with all-cause mortality, cardiovascular mortality, a higher risk of myocardial infarction, stroke and heart failure. Furthermore, FGF23 levels could induce left ventricular hypertrophy, potentially via direct but FGFR1c- and α-klotho-independent signaling in the heart.

  • Bone Diseases

    • Autosomal dominant hypophosphatemic rickets (ADHR)

    • Autosomal recessive hypophosphatemic rickets (ARHR)

    • X-linked hypophosphatemic rickets (XLH)

    • Autosomal recessive hypercholesterolaemia rickets (ARHR)

    • McCune-Albright syndrome/fibrous dysplasia

    • Hypophosphatemic rickets and hyperparathyroidism (HRHPT)

    • Drug-induced rickets/osteomalacia

    • Hyperphosphatemia

    • Hyperostosis-hyperphosphatemia syndrome

    • Fibrous dysplasia

    • Neurofibromatosis

    • Osteoglophonic dysplasia (OGD)

    • Linear nevus sebaceous syndrome (LNSS)/epidermal nevus syndrome (ENS)

    • Jansen-type metaphyseal chondrocdysplasia

  • Cancer

    • Tumor-induced osteomalacia

    • Familial tumor calcinosis

  • Cardiovascular disease

    • Heart failure

    • Left-ventricular hypertrophy

    • Vascular calcification

  • Endocrine disorders

    • Primary hyperparathyroidism

    • Secondary hyperparathyroidism

  • Kidney Disease

    • Chronic kidney disease (CKD)

    • Chronic kidney disease-mineral and bone disorder (CKD-MBD)

    • Chronic kidney disease and cardiovascular disease

    • Acute kidney injury

  • Metabolic disorders

    • Dyslipidemia

    • Insulin resistance/ type 2 diabetes

    • Anemia

Validation Data

 

All Biomedica ELISAs are validated according to FDA/ICH/EMEA guidelines. For more information about our validation guidelines, please refer to our quality page and published validation guidelines and literature.

Show validation literature

  • ICH Q2(R1) Validation of Analytical Procedures: Text and Methodology.

  • EMEA/CHMP/EWP/192217/2009 Guideline on bioanalytical method validation.

  • Bioanalytical Method Validation, Guidance for Industry, FDA, May 2018

Calibration

The FGF23 (C-terminal) immunoassay is calibrated against C-terminal FGF23 fragment (AA 180-251 of FGF23 (https://www.uniprot.org/uniprot/Q9GZV9)).

Detection Limit & Sensitivity

To determine the sensitivity of the FGF23 (C-terminal) ELISA, experiments measuring the lower limit of detection (LOD) and the lower limit of quantification (LLOQ) were conducted.

The LOD, also called the detection limit, is the lowest point at which a signal can be distinguished above the background signal, i.e. the signal that is measured in the absence of FGF23, with a confidence level of 99%. It is defined as the mean back calculated concentration of standard 1 (0 pmol/l of FGF23, five independent measurements) plus three times the standard deviation of the measurements.

The LLOQ, or sensitivity of an assay, is the lowest concentration at which an analyte can be accurately quantified. The criteria for accurate quantification at the LLOQ are an analyte recovery between 75 and 125% and a coefficient of variation (CV) of less than 25%. To determine the LLOQ, standard 2, i.e. the lowest standards containing FGF23, is diluted, measured five times and its concentration back calculated. The lowest dilution, which meets both criteria, is reported as the LLOQ.

The following values were determined for the FGF23 (C-terminal) ELISA:

LOD

0.08 pmol/l

LLOQ

0.1 pmol/l

Precision

The precision of an ELISA is defined as its ability to measure the same concentration consistently within the same experiments carried out by one operator (within-run precision or repeatability) and across several experiments using the same samples but conducted by several operators at different locations using different ELISA lots (in-between-run precision or reproducibility).

Within-Run Precision

Within-run precision was tested by measuring two samples six times within one FGF23 (C-terminal) ELISA lot. The experiment was conducted by one operator.

ID

n

Mean FGF23 [pmol/l]

SD [pmol/l]

CV [%]

Sample 1

6

0.6

0.07

12

Sample 2

6

10.0

0.6

6

In-Between-Run Precision

In-between-run precision was assessed by measuring two samples ten times within two different FGF23 (C-terminal) ELISA kit lots by four different operators.

 

n

Mean FGF23 [pmol/l]

SD [pmol/l]

CV [%]

Sample 1

10

0.61

0.06

10

Sample 2

10

9.91

0.5

5

Accuracy

The accuracy of an ELISA is defined as the precision with which it can recover samples of known concentrations.

The recovery of the FGF23 (C-terminal) ELISA was measured by adding recombinant C-terminal FGF23 fragment to samples containing a known concentration endogenous FGF23. The %recovery of the spiked concentration was calculated as the percentage of measured compared over the expected value.

This table shows the summary of the recovery experiments in the FGF23 (C-terminal) ELISA in different sample matrices:

   

% Recovery

Sample Matrix

n

+5 pmol/l

+10 pmol/l

Mean

Range

Mean

Range

Serum

13

96

84-108

89

60-103

EDTA plasma

7

97

73-123

94

81-114

Citrate plasma

7

100

61-132

90

58-107

Heparin plasma

8

101

68-161

92

69-118
Show individual measurements

Data showing recovery (R [%]) of recombinant C-terminal FGF23 fragment in human serum samples:

   

FGF23 [pmol/l]

% Recovery

  

Sample Matrix

ID

Reference

+5 pmol/l

+10 pmol/l

+5 pmol/l

+10 pmol/l

  

Serum

s1

1.3

6.4

10.1

103

87

  

Serum

s2

0.8

5.9

11.0

102

101

  

Serum

s3

2.2

7.0

10.4

96

82

  

Serum

s4

4.8

10.2

13.9

108

91

  

Serum

s5

2.1

7.1

12.4

100

103

  

Serum

s6

1.8

6.1

10.6

85

88

  

Serum

s7

1.2

5.5

7.3

84

60

  

Serum

s8

1.6

6.2

11.4

92

98

  

Serum

s9

2.7

7.2

11.7

90

90

  

Serum

s10

0.8

5.4

10.1

92

92

  

Serum

s11

1.1

5.9

10.4

95

93

  

Serum

s12

0.6

5.6

9.3

101

87

  

Serum

s13

1.9

7.1

10.4

104

85

  
         

96

89

Mean

         

84

60

Min
         

108

103

Max

Data showing recovery of recombinant C-terminal FGF23 fragment in human EDTA plasma samples:

   

FGF23 [pmol/l]

Recovery [%]

  

Sample Matrix

ID

Reference

+5 pmol/l

+10 pmol/l

+5 pmol/l

+10 pmol/l

  

EDTA plasma

e1

1.3

6.7

11.9

108

106

  

EDTA plasma

e2

1.4

5.0

9.5

73

81

  

EDTA plasma

e3

1.8

6.8

13.2

101

114

  

EDTA plasma

e4

1.2

5.4

8.9

84

77

  

EDTA plasma

e5

3.2

7.6

11.3

88

81

  

EDTA plasma

e6

2.0

7.0

11.6

100

96

  

EDTA plasma

e7

1.3

7.4

11.3

123

101

  
         

97

94

Mean

         

73

77

Min
         

123

114

Max

Data showing recovery of recombinant C-terminal FGF23 fragment in human citrate plasma samples:

   

FGF23 [pmol/l]

Recovery [%]

  

Sample Matrix

ID

Reference

+5 pmol/l

+10 pmol/l

+5 pmol/l

+10 pmol/l

  

Citrate plasma

c1

4.7

10.5

14.9

116

101

  

Citrate plasma

c2

2.2

8.8

12.0

132

98

  

Citrate plasma

c3

1.8

8.0

12.1

124

103

  

Citrate plasma

c4

1.8

7.2

12.5

110

107

  

Citrate plasma

c5

1.8

5.4

9.2

74

74

  

Citrate plasma

c6

2.7

5.8

8.6

61

58

  

Citrate plasma

c7

3.9

8.0

12.7

82

88

  
         

100

90

Mean

         

61

58

Min
         

132

107

Max

Data showing recovery of recombinant C-terminal FGF23 fragment in human heparin plasma samples:

   

FGF23 [pmol/l]

Recovery [%]

  

Sample Matrix

ID

Reference

+5 pmol/l

+10 pmol/l

+5 pmol/l

+10 pmol/l

  

Heparin plasma

c1

1.8

9.9

13.7

161

118

  

Heparin plasma

c2

2.2

7.1

11.5

97

93

  

Heparin plasma

c3

1.3

5.5

9.1

84

78

  

Heparin plasma

c4

2.2

7.1

11.7

98

95

  

Heparin plasma

c5

2.2

6.3

10.2

82

80

  

Heparin plasma

c6

1.2

4.6

8.1

68

69

  

Heparin plasma

c7

1.8

7.3

12.3

110

105

  

Heparin plasma

c8

2.2

7.5

12.1

106

99

  
         

101

92

Mean

         

68

78

Min
         

161

118

Max

Dilutuion Linearity & Paralellism

Tests of dilution linearity and parallelism ensure that both endogenous and recombinant samples containing FGF23 behave in a dose dependent manner and are not affected by matrix effects. Dilution linearity assesses the accuracy of measurements in diluted human samples spiked with known concentrations of recombinant analyte. By contrast, parallelism refers to dilution linearity in human samples and provides evidence that the endogenous analyte behaves in the same way as the recombinant one. Dilution linearity and parallelism are assessed for each sample type and should be within 20% of the expected concentration.

Parallelism was assessed by serially diluting clinical samples containing endogenous FGF23 with assay buffer.

The table below shows the mean recovery and range of serially diluted endogenous FGF23 in several sample matrices:

   

% Recovery of endogenous FGF23 in diluted samples

Sample

Matrix

n

1+1

1+3

1+7

Mean

Range

Mean

Range

Mean

Range

Serum

9

105

93-113

100

89-126

108

91-124

EDTA plasma

4

103

67-127

103

69-120

106

68-124

Citrate plasma

5

102

94-109

106

93-123

101

93-118

Heparin plasma

10

102

92-108

106

93-117

104

90-132
Show individual measurements

Data showing dilution linearity of endogenous FGF23 in human serum samples:

   

FGF23 [pmol/l]

% Recovery

  

Sample matrix

ID

Reference

1+1

1+3

1+7

1+1

1+3

1+7

  

Serum

s1

3.8

2.1

1.1

0.6

111

117

124

  

Serum

s2

16.2

8.1

3.6

1.9

99

89

92

  

Serum

s3

7.0

3.5

1.8

0.9

100

103

101

  

Serum

s4

9.0

4.6

2.1

1.0

102

95

91

  

Serum

s5

8.5

4.7

2.7

1.3

111

126

123

  

Serum

s6

6.0

3.4

1.3

0.9

113

90

121

  

Serum

s7

15.7

7.3

3.5

1.9

93

89

96

  

Serum

s8

20.4

10.9

5.1

2.6

106

101

103

  

Serum

s9

10.6

5.6

2.5

1.6

105

93

121

  
           

105

100

108

Mean

           

93

89

91

Min
           

113

126

124

Max

Data showing dilution linearity of endogenous FGF23 in human EDTA plasma samples:

   

FGF23 [pmol/l]

% Recovery

  

Sample Matrix

ID

Reference

1+1

1+3

1+7

1+1

1+3

1+7

  

EDTA plasma

e1

3.8

2.4

1.1

0.6

127

120

121

  

EDTA plasma

e2

23.3

13.6

6.7

3.2

117

115

112

  

EDTA plasma

e3

8.9

4.5

2.4

1.4

101

109

124

  

EDTA plasma

e4

20.7

7.0

3.6

1.8

67

69

68

  
           

103

103

106

Mean

           

67

69

68

Min
           

127

120

124

Max

Data showing recovery of endogenous FGF23 in a human citrate plasma sample:

   

FGF23 [pmol/l]

% Recovery

  

Sample Matrix

ID

Reference

1+1

1+3

1+7

1+1

1+3

1+7

  

Citrate plasma

e1

17.0

8.0

4.0

2.0

94

93

93

  

Citrate plasma

e2

13.0

7.1

4.0

1.9

109

123

118

  

Citrate plasma

e3

12.6

6.3

3.1

1.6

100

100

101

  

Citrate plasma

e4

10.0

5.3

2.6

1.2

106

106

97

  

Citrate plasma

e5

9.7

4.8

2.6

1.2

100

108

97

  
           

102

106

101

Mean

           

94

93

93

Min
           

109

123

118

Max

Data showing recovery of endogenous FGF23 in a human heparin plasma sample:

   

FGF23 [pmol/l]

% Recovery

  

Sample matrix

ID

Reference

1+1

1+3

1+7

1+1

1+3

1+7

  

Heparin plasma

s1

7.5

4.1

2.2

1.1

108

117

119

  

Heparin plasma

s2

5.8

2.9

1.7

1.0

102

117

132

  

Heparin plasma

s3

11.4

6.4

3.3

1.6

113

117

112

  

Heparin plasma

s4

9.0

4.9

2.6

1.3

113

117

112

  

Heparin plasma

s5

7.7

3.6

1.8

0.9

92

93

90

  

Heparin plasma

s6

10.7

5.3

2.6

1.2

100

97

91

  

Heparin plasma

s7

7.6

3.9

2.1

1.0

102

111

108

  

Heparin plasma

s8

6.4

3.2

1.5

0.8

99

93

95

  

Heparin plasma

s9

3.7

1.8

1.0

0.4

98

109

93

  

Heparin plasma

s10

8.6

4.1

2.0

1.0

96

95

90

  
           

102

106

104

Mean

           

92

93

91

Min
           

113

117

132

Max

Specificity

The specificity of an ELISA is defined as its ability to exclusively recognize the analyte of interest. The specificity of the FGF23 (C-terminal) ELISA was shown by characterizing both the capture and the detection antibodies through affinity measurements. In addition, the specificity of the ELISA was established through competition experiments, which measure the ability of the antibodies to exclusively bind to FGF23.

Isoforms

There are no isoforms of FGF23 known.

Affinities of Coating and Detection Antibodies

Antibody affinities to FGF23 were tested by biolayer interferometry measurements (Octet), which measures the binding of antibodies to a FGF23-coated sensor.

The results of these measurements are shown in the figure below.



Both antibodies used in the FGF23 (C-terminal) ELISA bind to FGF23 with high affinity.

Competition of Signal

Competition experiments were carried out by pre-incubating human samples with an excess of capture antibody. The concentration measured in this mixture was then compared to a reference value, which was obtained from the same sample but without the pre-incubation step. Mean competition was 95%.

     

FGF23 [pmol/l]

% Competition

  

Sample matrix

ID

Reference

Reference + capture AB

  

Serum

s1

2,7

0

100

  

Serum

s2

0,8

0,2

77

  

Serum

s3

5,6

0,1

98

  

Serum

s4

0,8

0

100

  

Serum

s5

0,5

0

100

  

Serum

s6

0,3

0

100

  

Serum

s7

0,3

0

100

  

EDTA plasma

e1

11,5

0,7

93

  

EDTA plasma

e2

8,5

0,5

94

  

EDTA plasma

e3

17,2

0,3

99

  

EDTA plasma

e4

8,9

0,2

98

  

Heparin Plasma

h1

10,5

0,4

96

  

Heparin plasma

h2

6,0

0,4

93

  

Heparin plasma

h3

5,3

0,2

96

  

Heparin plasma

h4

3,1

0,4

88

  
       

95

Mean

Sample Stability

The stability of endogenous C-terminal FGF23 fragment was tested by comparing five measurements in samples that had undergone four freeze-thaw cycles.

For freeze-thaw experiments, samples were collected according to the supplier’s instruction using blood collection devices and stored at -80°C. Reference samples were freeze-thawed once. The mean recovery of sample concentration after four freeze-thaw cycles is 101%.

   

FGF23 [pmol/l]

 % Recovery after 4 freeze/thaw cycles  

Sample matrix

ID

Reference

1x

2x

3x

4x

  

Serum

s1

16,9

15,6

16,7

16,6

16,3

96%

  

Serum

s2

5,0

5,0

5,5

5,7

6,8

136%

  

Serum

s3

12,6

11,4

14,2

11,2

11,7

93%

  

Serum

s4

13,9

12,9

14,1

13,0

13,3

96%

  

Serum

s5

12,7

11,6

13,9

12,6

11,7

92%

  

Serum

s6

6,3

6,2

6,3

5,8

5,7

91%

  
             

101

Mean

All samples should undergo a maximum of four freeze-thaw cycles.

Sample Values

FGF23 Values in Apparently Healthy Individuals

To provide expected values for circulating FGF23, a panel of samples from apparently healthy donors was tested.

A summary of the results is shown below:

   

FGF23 (C-terminal) [pmol/l]

Sample Matrix

n

Mean

Median

5% Percentile

95% Percentile

Minimum

Maximum

Serum

35

1.1

0.8*

0.3

3.0

0.2

4.2

EDTA plasma

22

1.6

1.3

0.6

4.0

0.3

4.8

Heparin plasma

22

1.5

1.2

0.5

3.8

0.3

4.0

Citrate plasma

30

1.9

1.4**

0.9

4.5

0.7

6.8

* Serum and plasma values of FGF23 (C-terminal) show a correlation of R2= 0.8675 (see matrix comparison data).

**Various plasma matrices (EDTA, heparin, citrate) show a correlation of R2= 0.9535 (see matrix comparison data).

It is recommended to establish the normal range for each laboratory.

FGF23 (C-terminal) Values in Chronic Kidney Disease and Dialysis Panels

In addition to samples from apparently healthy donors, several panels of samples from chronic kidney disease (CKD) and dialysis patients were tested.

Summary of the results obtained with several chronic kidney disease (dialysis) panels:

     

FGF23 (C-terminal) [pmol/l]

Sample Matrix

n

Panel

Mean

Median

5% Percentile

95% Percentile

Minimum

Maximum

Serum

18

CKD

8.2

5.7

1.4

20

1.4

20.0

Serum

20

Dialysis

89

59

6

307

6

310

Serum

15

Dialysis

96

20

3

408

3

408

Plasma

20

Dialysis

248

177

24

717

24

725

Heparin plasma

18

Dialysis

441

355

150

882

150

882

Comparison of FGF23 (C-terminal) Values in Apparently Healthy Individuals and Patients with Chronic Kidney Disease (CKD)/ Dialysis

Matrix Comparison

To assess whether all tested matrices behave the same way in the FGF23 (C-terminal) ELISA, concentrations of FGF23 were measured in serum, EDTA, heparin and citrate plasma samples prepared from 22 apparently healthy donors. Each individual donated blood in all tested sample matrices. In addition, FGF23 concentrations were assessed in serum and plasma and plasma samples prepared from 20 patients with chronic kidney disease (dialysis). Correlations between serum and plasma samples were calculated for each cohort.

Correlations of FGF23 measurements in serum and plasma samples is shown below:


 



Both serum and plasma can be measured with the FGF23 (C-terminal) ELISA.

Comparison With Other FGF23 (C-terminal) ELISA Assays

The FGF23 (C-terminal) ELISA (cat. no. BI-20702) was compared with the FGF23 C-Term ELISA (cat. no. 60-6100) manufactured by Immutopics. The same panel of samples, consisting of EDTA plasma samples from apparently healthy donors (n=33) and patients with chronic kidney disease (dialysis) (n=20), was tested. The correlation between the two assays was R2=0.9295.

The figure below shows the measured concentrations in both tests:



Citations

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