1Human KL-6 ELISA
MATERIAL REQUIRED BUT NOT SUPPLIED

Cat. No.: BA1017
Enzyme Immunoassay for the quantitative determination of KL-6 in humanserum, plasma and bronchoalveolar lavage fluid (BALF).
Interstitial lung diseases (ILD) are a group of lung disorders characterized byinterstitial lung thickening. Krebs von den Lungen-6 (KL-6) is a 200 kDa
glycoprotein that is predominantly expressed by damaged alveolar type II
cells, and it has been proposed as a potential biomarker of different ILD. 1KL-6 has profibrotic and anti-apoptotic effects on lung fibroblasts andreflects the degree and severity of alveolar epithelial injury. KL-6 ispredictive biomarker useful in the clinical management of ILD patients,
particularly in patients with severe fibrotic lung disorders.1
Serum KL-6 levels were also found elevated in Rheumatoid Arthritis –Associated IDL patients, and the levels positively correlated with HRCTfibrosis score.2
PRINCIPLE OF KL-6 ELISA

1.
2.
3.
4.
5.

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

The microtiter plate is coated with the antibody specifically binding theKL-6. The human serum, plasma or BALF is incubated in the plate with thecapture 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 KL-6 in the specimen. The
concentration of KL-6 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 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 KL-6 Master Standard in Dilution Buffer, forthe volume information see the Certificate of Analysis. Let it rehydrate for
15 min The concentration of human KL-6 in Master Standard is
10 U/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
ItemAntibody Coated Microtiter Plate
Biotin-labelled Antibody
Streptavidin-HRP Conjugate
Master Standard
Quality Control A (human serum, lyophilized)
Quality Control B (human serum, lyophilized)
Dilution Buffer
Wash Buffer 15x conc.
Substrate Solution
STOP Solution

v_01

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

Std1

Standard 10 U/mL (lyophilized)

Dilution
BufferSee CofA

Concentration

Std2

300 µL of Std1

300 µL

5 U/mL

Std3300 µL of Std2

300 µL

2.5 U/mL

Std4300 µL of Std3300 µL

1.25 U/mL

Std5300 µL of Std4300 µL

0.625 U/mL

Std6300 µL of Std5300 µL

0.156 U/mLBlank-300 µL0 U/mL

10 U/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 1:200 prior touse, see Preparation of samples.

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 at
4oC for two weeks or at -20oC for long term storage.

Dilution linearity
SamplePreparation of samples

Measuredconcentration(U/mL)
629
309
155
78
323
159
81
44

Expected
concentration(U/mL)
314
157
79
162
81
40

Yield(%)

Measuredconcentration(U/mL)
195
737
440
266

Expected
concentration(U/mL)
695
445
257

Yield(%)

2x4x8x22x4x8x98
98
100
99
100
110

Spiking Recovery
SampleSpike(U/mL)

1500
250
62.5

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 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 or 450/630 nm within 15 min.

Dilution1

Human serum, plasma or BALF 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 1:200. It is recommended to use thetwo-step dilution.
Dilution A (10x) for both singlets and duplicates: 5 µL of samples + 45 µL ofDilution Buffer.
Dilution B (20x): 8 µL of Dilution A + 152 µL of Dilution Buffer, for singlets;
15 µL of Dilution A + 285 µL of Dilution Buffer for duplicates.
Do not store the diluted samples.

Accuracy

1.
2.

106
99
103

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.

Human KL-6 Standard Curve
43.5
3Absorbance (450/630 nm)

PERFORMANCE CHARACTERISTICS
Samples used in the tests were diluted 1:200 as recommended and assayed.
The results are multiplied by the dilution factor.
1.

Sensitivity

The limit of detection, defined as a concentration of human KL-6 giving
absorbance higher than absorbance of blank + 3 standard deviations, isbetter than 0.04 U/mL of sample.
2.

v_01

1.5
10
Mean (U/mL)
631
193

SD
13.6
3.9CV (%)
2.2
2.0

Mean (U/mL)
526
185

5

10204080160

Concentration (U/mL)

RESOURCE
1Inter-assay (Run – to – run)
Sample122

0.5Precision

Intra-assay
Sample
122.5

SD
11.2
3.7

CV (%)
2.1
2.0

Prisco D, Grifoni E. The role of KL-6 testing in patients with suspected venous
thromboembolism. Semin Thromb Hemost. 2009 Feb;35(1):50-9. doi:
10.1055/s-0029-1214148. Epub 2009 Mar 23. PMID: 19308893.
2WELLS, P.S. (2007), Integrated strategies for the diagnosis of venous
thromboembolism. Journal of Thrombosis and Haemostasis, 5: 41-50.
https://doi.org/10.1111/j.1538-7836.2007.02493.x