1Human Retinol-binding Protein 4 ELISA
MATERIAL REQUIRED BUT NOT SUPPLIED

Cat. No.: BA1004
Enzyme Immunoassay for the quantitative determination of Retinol-bindingProtein 4 (RBP4) in human serum and plasma.
Retinol-binding Protein 4 (RBP4) is a member of the lipocalin family and themajor transport protein of the hydrophobic molecule retinol, also known asvitamin A, in the circulation.1 Retinol-binding Protein 4 (RBP4) is elevated inserum and adipose tissue (AT) in obesity-induced insulin resistance andcorrelates inversely with insulin-stimulated glucose disposal. Serum RBP4levels correlated inversely with glucose disposal and insulin-mediatedsuppression of lipolysis, FFA, and EGP.2
High levels of serum RBP4 are associated with chronic kidney disease (CKD)
and urine BRP4 is the most sensitive biomarker of proximal renal tubule
function.
PRINCIPLE OF RBP4 ELISA

1.
2.
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4.
5.

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

The microtiter plate is coated with the antibody specifically binding theRetinol-binding Protein 4. The human serum or 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 RBP4 in the specimen. Theconcentration of RBP4 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

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 labwareand 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 RBP4 Master Standard in Dilution Buffer,
for the volume information see the Certificate of Analysis. Let it rehydrate
for 15 min. The concentration of human RBP4 in Master Standard is
120 ng/mL.
Prepare set of Standard solution as follows:
Use the Master Standard to produce a dilution series (as below). Mix eachtube thoroughly before the next transfer. The Dilution Buffer serves asBlank.
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_02

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

Std1

Standard 100 ng/mL (lyophilised)

Dilution
Buffer1000 µL

Std2

300 µL of Std1

300 µL

60 ng/mL

Std3300 µL of Std2300 µL

30 ng/mL

Std4300 µL of Std3300 µL

15 ng/mL

Std5300 µL of Std4300 µL

7.5 ng/mL

Std6Blank

300 µL of Std5300 µL
300 µL

3.75 ng/mL
0 ng/mL-Concentration
120 ng/mL

Preparation of Quality Control A and B
Reconstitute the lyophilized human serum Quality Controls indeionized/distilled, for the volume information see the Certificate ofAnalysis. Let the QCs rehydrate for 15 min and dilute them 1:750 in Dilution
Buffer, prior to use, see Preparation of samples (use the two-step dilution).

2

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.

Dilution linearity
SamplePreparation of samples

11.

Sensitivity

The limit of detection, defined as a concentration of human RBP4 giving
absorbance higher than absorbance of blank + 3 standard deviations, isbetter than 0.94 ng/mL of sample.
2.

Yield
(%)
8993114The 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 standardpoints properly. The concentrations need to be multiplied by the dilution
factor, either automatically by reader or manually.

Human RBP4 Standard Curve
3.5
3
2.5
2
1.5
1
0.5

Precision

0Intra-assay
Sample12Expected
concentration(µg/mL)
28.5
23.8
21.4

Typical standard curve

Absorbance (450/630 nm)

Samples used in the tests were diluted 1:750 as recommended and assayed.
The results are multiplied by dilution factor.

Measuredconcentration(µg/mL)
19.1
25.4
22.1
24.4

8185
1088488110

Spiking Recovery

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2.

PERFORMANCE CHARACTERISTICS

Spike(µg/mL)+ 9.4+ 4.7+2.32

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 for30 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 10 min,at 25oC ± 2oC. Avoid exposure to the light during this step.
9. Pipette 100 µL of STOP solution.
10. Read the signal at 450/630 nm within 15 min.

Expected
concentration(µg/mL)
11.2
5.6
2.8
11.5
5.82.9Yield(%)

2x4x8x

Measuredconcentration(µg/mL)
22.4
9.1
4.8
3.0
23.0
9.7
5.1
3.2

2x4x8xSample

Do not store the diluted samples.

Dilution1

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.
Recommended dilution of samples is 1:750. It is recommended to use thetwo-step dilution.
Dilution A (25x) for both singlets and duplicates: 5 µL of samples + 120 µL ofDilution Buffer.
Dilution B (30x): 5 µL of Dilution A + 145 µL of Dilution Buffer, for singlets;
9 µL of Dilution A + 261 µL of Dilution Buffer for duplicates.

Accuracy

Mean (µg/mL)
24.5
22.1

SD
2.8
2.6

CV (%)
1212

3.75

7.5

1530

60

120

Concentration (ng/mL)
RESOURCES
1 Steinhoff JS, Lass A, Schupp M. Biological Functions of RBP4 and Its Relevance for

Human
Diseases.
Front
Physiol.
2021
Mar11;12:659977.
10.3389/fphys.2021.659977. PMID: 33790810; PMCID: PMC8006376.

Inter-assay (Run – to – run)
Sample12v_02

Mean (µg/mL)
40.5
35.7

SD
4.2
1.8CV (%)
105

doi:

2 Kilicarslan M, de Weijer BA, Simonyté Sjödin K, Aryal P, Ter Horst KW, Cakir H, Romijn

JA, Ackermans MT, Janssen IM, Berends FJ, van de Laar AW, Houdijk AP, Kahn BB, Serlie
MJ. RBP4 increases lipolysis in human adipocytes and is associated with increased
lipolysis and hepatic insulin resistance in obese women. FASEB J. 2020 May;34(5):60996110. doi: 10.1096/fj.201901979RR. Epub 2020 Mar 13. PMID: 32167208; PMCID:
PMC7317205.