1

Human Insulin-like Growth Factor-binding
Protein 7 (IGFBP7) ELISA
Cat. No.: BA1024
Enzyme Immunoassay for the quantitative determination Insulin-likeGrowth Factor-binding Protein 7 in human serum, plasma, and urine.
IGFBP7 is a member of the insulin superfamily of growth promoting
peptides, and is one of the most abundant and common growth factor
polypeptides. IGFBPs are a class of secreted proteins that is able to interact
with many ligands other than IGFs.1
Currently, IGFBP-7 has been used as an early diagnosis and prognostic
marker for acute renal insufficiency (AKI). IGFBP-7, and TIMP-2
concentrations in the urine of children after receiving injections of contrast
medium increased faster than SCr. The combination of IGFBP-7 and TIMP-2
was better than either analyte alone (the specificity was 80.0%, and thesensitivity was 81.2%).2 Based on the results of two studies, the urine
compound of TIMP-2 and IGFBP-7 became US Food and Drug Administration(FDA)-approved biomarker for risk assessment of AKI in ICU patients.1IGFBP-7 has also been proposed as a potential prognostic biomarker inheart failure (HF). It provided prognostic information incremental to clinicalvariables, NT-proBNP, and hsTnT.3
PRINCIPLE OF INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN
7 ELISAThe microtiter plate is coated with the antibody specifically binding Insulinlike Growth Factor-binding Protein 7. The human serum, plasma or urine is
incubated in the plate with the capture antibody.
The specimen is washed out and the specifically bound protein is incubatedwith HRP-labelled detection antibody. Unbound reagent is then washedout. Horseradish peroxidase (HRP) bound in the complex reacts with thechromogenic substrate (TMB) creating the blue colour. The reaction isstopped 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 the Insulin-like Growth Factorbinding Protein 7 the specimen. The concentration of Insulin-like Growth
Factor-binding Protein 7 in unknown samples is determined from thecalibration curve which is created by plotting the absorbance values against
the standard concentration values.

Kit Contents
ItemAntibody 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

Qty.
96 wells
13 mL
1 vial1 vial1 vial2x13 mL
50 mL
13 mL
13 mL

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

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

WARNINGS AND PRECAUTIONS
1.
2.
3.
4.
5.
6.
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 IGFBP7 Standard in Dilution Buffer, for thevolume information see the Certificate of Analysis. Let it rehydrate for 15
min prior to use. The concentration of human IGFBP7 in Master Standard is
4 ng/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_01

Std1

Standard 4 ng/mL (lyophilized)

Dilution
BufferSee CofA

Concentration

Std2

250 µL of Std1

250 µL

2 ng/mL

Std3

250 µL of Std2

250 µL

1 ng/mL

Std4

250 µL of Std3

250 µL

0.5 ng/mL

Std5

250 µL of Std4

250 µL0.25 ng/mL

Std6250 µL of Std5

250 µL

0.125 ng/mLBlank-200 µL

0 ng/mL4 ng/mL

2Preparation of Quality Control A and B3.

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 1:80 in Dilution
Buffer, prior to use, see Preparation of samples.

Accuracy

Dilution linearity
SamplePreparation of Wash Buffer 1x1Prepare 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.

2Dilution

Expectedconcentration(ng/mL)
42.9
21.4
10.7
46.7
23.3
11.7

Yield(%)

Measuredconcentration(ng/mL)
68.8
250.2
162.4
121.9

Expected
concentration(ng/mL)
228.8
148.8
108.8

Yield (%)

2x4x8x2x4x8x

Preparation of samples
Human serum, plasma or ur1ne 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.

Measuredconcentration(ng/mL)
85.8
41.0
17.7
8.56
93.4
43.5
20.0
9.4968380
938680

Spiking Recovery

Recommended dilution for serum, plasma and urine 1:80. It is
recommended to use the two-step dilution.
Dilution A (8x) for both singlets and duplicates: 10 µL of samples + 70 µL ofDilution Buffer.
Dilution B (10x): 20 µL of Dilution A + 180 µL of Dilution Buffer, for singlets;
30 µL of Dilution A + 270 µL of Dilution Buffer, for duplicates.
Do not store the diluted samples.

SampleSpike(ng/ml)

1

160
80
40109
109
112

ASSAY PROCEDURE

3.

4.
5.
6.
7.
8.

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 for2 hours at 25oC ±2oC, shaking at 300 rpm.
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.
Pipette 100 µL of HRP-labelled Antibody Conjugate into eachwell. Incubate for 1 hour at 25oC ±2oC, shaking at 300 rpm.
Wash the wells as described in point 3.
Pipette 100 µL Substrate solution, incubate for 10 min,at 25oC ±2oC. Avoid exposure to the light during this step.
Pipette 100 µL of STOP solution.
Read the signal at 450 or 450/630 nm within 10 min.

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.

3.53PERFORMANCE CHARACTERISTICS
Samples used in the tests were diluted 1:80 as recommended and assayed.
The results are multiplied by the dilution factor.
1.

Sensitivity

The limit of detection, defined as a concentration of human
IGFBP7 giving absorbance higher than absorbance of blank + 3 standard
deviations, is better than 0.031 ng/mL of sample.
2.

2.5
2
1.5
10.5

Precision

00.125Intra-assay
Sample12

Human IGFBP 7 Standard Curve

4Absorbance (450/630 nm)

1.
2.

Mean (ng/mL)
84.2
90.0

SD
4.2
4.3

CV (%)
55

0.250.5124Concentration (ng/mL)

RESOURCES
1Wang S, Chi K, Wu D, Hong Q. Insulin-Like Growth Factor Binding Proteins in Kidney Disease. Front
Pharmacol. 2021 Dec 22;12:807119. doi: 10.3389/fphar.2021.807119. PMID: 35002740; PMCID:
PMC8728008.

Inter-assay (Run – to – run)
Sample12Mean (ng/mL)
77.6
80.4

SD
3.7
8.3

CV (%)
5102

Sun Q, Kang Z, Li Z, Xun M. Urinary NGAL, IGFBP-7, and TIMP-2: novel biomarkers to predict contrast
medium-induced acute kidney injury in children. Ren Fail. 2022 Dec;44(1):1201-1206. doi:
10.1080/0886022X.2022.2075277. PMID: 36120960; PMCID: PMC9518296.
3

Adamson C, Welsh P, Docherty KF, de Boer RA, Diez M, Drożdż J, Dukát A, Inzucchi SE, Køber L,Kosiborod MN, Ljungman CEA, Martinez FA, Ponikowski P, Sabatine MS, Morrow DA, Lindholm D,Hammarstedt A, Boulton DW, Greasley PJ, Langkilde AM, Solomon SD, Sattar N, McMurray JJV, JhundPS. IGFBP-7 and Outcomes in Heart Failure With Reduced Ejection Fraction: Findings From DAPA-HF.
JACC Heart Fail. 2022 Oct 20:S2213-1779(22)00571-6. doi: 10.1016/j.jchf.2022.09.004. Epub aheadof print. PMID: 36592046.

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