1

Human Growth differentiation factor 15
ELISACat. No.: BA1025
Enzyme Immunoassay for the quantitative determination of Growthdifferentiation factor 15 (GDF15) in human serum and plasma.
Growth differentiation factor 15 (GDF15) is a member of the TGFβ
superfamily whose expression is increased in response to cellular stress anddisease as well as by metformin.1 Identified as a new heart-derived
endocrine hormone that regulates body growth, GDF15 has a local
cardioprotective role, presumably due to its autocrine/paracrine
properties: antioxidative, anti-inflammatory, antiapoptotic. GDF15
expression is highly induced in cardiomyocytes after ischemia/reperfusion
and in the heart within hours after myocardial infarction (MI). GDF15 may
be a predictive biomarker of adverse cardiac events.2 Available evidence
also suggests that a substantial amount of GDF15 is secreted in various
human cancers, such as ovarian cancer, prostate cancer, and breast cancer,
among others.3

MATERIAL REQUIRED BUT NOT SUPPLIED
1.
2.
3.
4.
5.

WARNINGS AND PRECAUTIONS
1.
2.
3.
4.
5.
6.
7.

PRINCIPLE OF GDF15 ELISA
The microtiter plate is coated with the antibody specifically binding theGrowth differentiation factor 15. The human serum or plasma is incubatedin the plate 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 GDF15 in the specimen. The
concentration of GDF15 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

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 GDF15 Standard in Dilution Buffer, for thevolume information see the Certificate of Analysis. Let it rehydrate for
15 min. The concentration of human GDF15 in Master Standard is
800 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

v_02

Standard 800 pg/mL (lyophilized)

Std2300 µL of Std1300 µL

400 pg/mL

Std3300 µL of Std2300 µL

200 pg/mL

Std4300 µL of Std3300 µL

100 pg/mL

Std5300 µL of Std4300 µL

50 pg/mL

Std6300 µL of Std5300 µL

25 pg/mL-300 µL

0 pg/mLBlank

Kit Contents
Item
Antibody Coated Microtiter Plate
Antibody-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

Std1

Dilution
BufferSee CofAQty.
96 wells
13 mL
1 vial1 vial1 vial2x13 mL
50 mL
13 mL
13 mL

Concentration
800 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 1:10 in Dilution
Buffer, prior to use, see Preparation of samples.

23.

Preparation of Wash Buffer 1x
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
SampleDilution

Measuredconcentration(pg/mL)
1818
825
415
186
2871
1460
711
348

Expected
concentration(pg/mL)
909
454
227
1436
718
359

Yield(%)

Measuredconcentration(pg/mL)
33
142
8960

Expected
concentration(pg/mL)
133
83
58

Yield (%)

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

2x4x8x2

Recommended dilution of samples is 1:10 for healthy individuals, i.e., forsinglets 20 µL of sample + 180 µL of Dilution Buffer, for duplicates 30 µL ofsamples + 270 µL of Dilution Buffer, respectively.
Recommended dilution of samples is 1:40 for individuals in condition inwhich is expected higher level of GDF15, i.e., for singlets 5 µL of sample +195 µL of Dilution Buffer, for duplicates 10 µL of samples + 390 µL of Dilution
Buffer, respectively.

2x4x8xSpiking Recovery
SampleSpike(pg/ml)

11005025Do not store the diluted samples.
ASSAY PROCEDURE
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 for
1 hour at 25oC ±2oC, shaking at 300 rpm.
3. Wash the wells 3-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 1 hour 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 20 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.

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 GDF15 Standard Curve
3.5
3

PERFORMANCE CHARACTERISTICS
Samples used in the tests were diluted 1:10 as recommended and assayed.
The results are multiplied by the dilution factor.
1.

Sensitivity

2.5
2
1.5
10.5

The limit of detection, defined as a concentration of human GDF15 giving
absorbance higher than absorbance of blank + 3 standard deviations, isbetter than 6 pg/mL of sample.
2.

025Precision

Mean (pg/mL)
1649
1696

SD
158
54

CV (%)
103

SD
30
61

CV (%)
44Inter-assay (Run – to – run)
Sample
1
2

v_02

50

100

200

400

800

Concentration (pg/mL)

Intra-assay
Sample
12

107
107
103

Typical standard curve

Absorbance (450/630 nm)

1.
2.

91
91
82
102
99
97

Mean (pg/mL)
841
1604

RESOURCES1Wang D, Day EA, Townsend LK, Djordjevic D, Jørgensen SB, Steinberg GR. GDF15: emerging biology
and therapeutic applications for obesity and cardiometabolic disease. Nat Rev Endocrinol. 2021
Oct;17(10):592-607. doi: 10.1038/s41574-021-00529-7. Epub 2021 Aug 11. PMID: 34381196.
2
Rochette L, Dogon G, Zeller M, Cottin Y, Vergely C. GDF15 and Cardiac Cells: Current Concepts andNew Insights. Int J Mol Sci. 2021 Aug 18;22(16):8889. doi: 10.3390/ijms22168889. PMID: 34445593;
PMCID: PMC8396208.
3Li S, Ma YM, Zheng PS, Zhang P. GDF15 promotes the proliferation of cervical cancer cells byphosphorylating AKT1 and Erk1/2 through the receptor ErbB2. J Exp Clin Cancer Res. 2018 Apr
10;37(1):80. doi: 10.1186/s13046-018-0744-0. PMID: 29636108; PMCID: PMC5894198.