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Human Galectin-3 ELISA

MATERIAL REQUIRED BUT NOT SUPPLIED

Cat. No.: BA1007
Enzyme Immunoassay for the quantitative determination Galectin-3 inhuman serum and plasma.
Galectin-3 is a member of the galectin family, which are β-galactosidebinding lectins with ≥1 evolutionary conserved carbohydrate-recognition
domain.1 Galectin-3 is expressed by various immune cells and is readily
secreted by injured and inflammatory cells. Thus, galectin-3 is a novel
candidate biomarker for the diagnosis, analysis, and prognosis of various
cardiac diseases, including heart failure.2
Gallectin-3 has also been linked to kidney disease in clinical studies, and its
inhibition appears to improve renal disease.
PRINCIPLE OF GALECTIN-3 ELISA

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WARNINGS AND PRECAUTIONS
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The microtiter plate is coated with the antibody specifically binding theGalectin-3. The human serum or plasma is incubated in the plate with thecapture 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 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 Galectin-3 in the specimen.
The concentration of Galectin-3 in unknown samples is determined fromthe calibration 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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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
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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 Galectin-3 Standard in Dilution Buffer, forthe volume information see the Certificate of Analysis. Let it rehydrate for
15 min. The concentration of human Galectin-3 in Master Standard is
4 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
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
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Qty.
96 wells
13 mL
1 vial1 vial1 vial
13 mL
50 mL
13 mL
13 mL

Std1

Standard 4 ng/mL (lyophilised)

Dilution
Buffer500 µL

Concentration

Std2

250 µL of Std1

250 µL

Std3

250 µL of Std2

250 µL

2 ng/mL
1 ng/mL

Std4

250 µL of Std3

250 µL

0.5 ng/mL

Std5250 µL of Std4

250 µL0.25 ng/mL

Std6250 µL of Std5

250 µL

0.125 ng/mL

Blank-250 µL

0 ng/mL

4 ng/mL

Preparation of Quality Control A and B
Reconstitute the lyophilized human serum Quality Controls indeionized/distilled water, for the volume information see the Certificate ofAnalysis. Let the QCs rehydrate for 15 min and dilute them 1:20 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 at
4oC for two weeks or at -20oC for long term storage.
Preparation of samples
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 1:20, i.e., 7 µL of sample + 133 µL ofDilution Buffer for singlets and 14 µL of sample + 266 µL of Dilution Buffer
for duplicates.

Accuracy

Dilution linearity
SampleDilution

12x4x8x22x4x8x

Measuredconcentration(ng/mL)
8.5
4.4
2.5
1.3
9.9
4.9
2.7
1.4

Expected
concentration(ng/mL)
4.3
2.1
1.1
5.0
2.5
1.2

Yield(%)

Measuredconcentration(ng/mL)
9.0
30.2
18.6
11.6

Expected
concentration(ng/mL)
29.0
19.0
13.0

Yield(%)

10411611999108113

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 for
1 hour 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 15 min.

SampleSpike(ng/mL)

120104

1049890

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.

PERFORMANCE CHARACTERISTICS
Samples used in the tests were diluted 1:20 as recommended and assayed.
The results are multiplied by the dilution factor.
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Sensitivity

The limit of detection, defined as a concentration of human Galectin-3
giving absorbance higher than absorbance of blank + 3 standard deviations,is better than 0.03 ng/mL of sample.
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Precision

Intra-assay
Sample12

Mean (ng/mL)
8.6
10.4

SD0.4
0.5

CV (%)
55SD
0.40.3CV (%)
93Inter-assay (Run – to – run)
Sample12Mean (ng/mL)
4.9
9.0

RESOURCES
1 Dong R, Zhang M, Hu Q, Zheng S, Soh A, Zheng Y, Yuan H. Galectin-3 as a novel

biomarker for disease diagnosis and a target for therapy (Review). Int J Mol Med. 2018
Feb;41(2):599-614. doi: 10.3892/ijmm.2017.3311. Epub 2017 Dec 5. PMID: 29207027;
PMCID: PMC5752178.
2 Hara, A.; Niwa, M.; Kanayama, T.; Noguchi, K.; Niwa, A.; Matsuo, M.; Kuroda, T.;

Hatano, Y.; Okada, H.; Tomita, H. Galectin-3: A Potential Prognostic and Diagnostic
Marker for Heart Disease and Detection of Early Stage Pathology. Biomolecules 2020,
10, 1277. https://doi.org/10.3390/biom10091277

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