Instructions for use

Kynurenine ELISABA E-2200R

Kynurenine ELISA
1. Introduction
1.1 Intended use and principle of the test
Enzyme Immunoassay for the quantitative determination of L-Kynurenine in serum, plasma and various
biological samples.
After acylation Kynurenine is quantitatively determined by ELISA.
The competitive ELISA uses the microtiter plate format. The antigen is bound to the solid phase of themicrotiter plate. The acylated standards, controls and samples and the solid phase bound analyte compete
for a fixed number of antibody binding sites. When the system is in equilibrium, free antigen and freeantigen-antibody complexes are removed by washing. The antibody bound to the solid phase is detected byan anti-rabbit IgG-peroxidase conjugate using TMB as a substrate. The reaction is monitored at 450 nm.
Quantification of unknown samples is achieved by comparing their absorbance with a reference curve
prepared with known standards.
1.2 BackgroundKynurenine is a non-proteinogenic amino acid that is produced as a metabolic intermediate during thedegradation of tryptophan. The degradation of tryptophan is catalyzed by the inducible enzyme indolamine2,3-dioxygenase (IDO). The product is kynurenine. Cytokines, in particular interferon-γ, influence theactivity of the IDO, so that’s why the kynurenine path is closely linked to the immune system. Kynurenine
can be further converted to neuroprotective kynurenic acid, but also to neurotoxic quinolinic acid.
2. Procedural cautions, guidelines, warnings and limitations
2.1 Procedural cautions, guidelines and warnings(1) This kit is intended for professional use only. Users should have a thorough understanding of this protocol
for the successful use of this kit. Only the test instruction provided with the kit is valid and has to be used
to run the assay. Reliable performance will only be attained by strict and careful adherence to theinstructions provided.
(2) This assay was validated for a certain type of sample as indicated in Intended Use (please refer toChapter 1). Any off-label use of this kit is in the responsibility of the user and the manufacturer cannot beheld liable.
(3) The principles of Good Laboratory Practice (GLP) have to be followed.
(4) In order to reduce exposure to potentially harmful substances, wear lab coats, disposable protective
gloves and protective glasses where necessary.
(5) All kit reagents and specimens should be brought to room temperature and mixed gently but thoroughly
before use. Avoid repeated freezing and thawing of reagents and specimens.
(6) For dilution or reconstitution purposes, use deionized, distilled, or ultra-pure water.
(7) The microplate contains snap-off strips. Unused wells must be stored at 2 °C to 8 °C in the sealed foil
pouch with desiccant and used in the frame provided.
(8) Duplicate determination of sample is highly recommended to be able to identify potential pipetting errors.
(9) Once the test has been started, all steps should be completed without interruption. Make sure that therequired reagents, materials and devices are prepared ready at the appropriate time.
(10) Incubation times do influence the results. All wells should be handled in the same order and timeintervals.
(11) To avoid cross-contamination of reagents, use new disposable pipette tips for dispensing each reagent,sample, standard and control.
(12) A standard curve must be established for each run.
(13) The controls should be included in each run and fall within established confidence limits. The confidence
limits are listed in the QC-Report.
(14) Do not mix kit components with different lot numbers within a test and do not use reagents beyond
expiry date as shown on the kit labels.
(15) Avoid contact with Stop Solution containing 0.25 M H2SO4. It may cause skin irritation and burns. In caseof contact with eyes or skin, rinse off immediately with water.
(16) TMB substrate has an irritant effect on skin and mucosa. In case of possible contact, wash eyes with anabundant volume of water and skin with soap and abundant water. Wash contaminated objects before
reusing them.
(17) For information on hazardous substances included in the kit please refer to Safety Data Sheet (SDS). The
Safety Data Sheet for this product is made available directly on the website of the manufacturer or uponrequest.
(18) Kit reagents must be regarded as hazardous waste and disposed of according to national regulations.
2.2 Limitations
Any inappropriate handling of samples or modification of this test might influence the results.
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2.2.1 Interfering substances
Serum/Plasma
Samples containing precipitates or fibrin strands might cause inaccurate results. Hemolytic samples (up to 4
mg/ml hemoglobin), icteric samples (up to 50 mg/dl bilirubin) and lipemic samples (up to 1700 mg/dltriglycerides) have no influence on the assay results.
2.2.2 Drug interferences
There are no known substances (drugs) which ingestion interferes with the measurement of kynurenine levelin the sample.
2.2.3 High-Dose-Hook effectNo hook effect was observed in this test.
3. Storage and stability
Store the unopened reagents at 2 - 8 C until expiration date. Do not use components beyond the expiry
date indicated on the kit labels. Once opened the reagents are stable for 1 month when stored at
2 – 8 °C. Once the resealable pouch has been opened, care should be taken to close it tightly with desiccantagain.
4. Materials
4.1 Content of the kit
BA D-0032
Content:

Microtiter Plate - Ready to use
1 x 96 well plate, empty
Adhesive Foil - Ready to use

BA D-0090
Content:

Adhesive Foils in a resealable pouch

Volume:1 x 4 foils

BA E-0030

Wash Buffer Concentrate - Concentrated 50x

Content:

Buffer with a non-ionic detergent and physiological pH

Volume:1 x 20 ml/vial, light purple cap

BA E-0040

Enzyme Conjugate - Ready to use

Content:

Goat anti-rabbit immunoglobulins conjugated with peroxidase

Volume:1 x 12 ml/vial, red cap

BA E-0055

Substrate - Ready to use

Content:

Chromogenic substrate containing tetramethylbenzidine, substrate buffer and hydrogenperoxide

Volume:1 x 12 ml/vial, black cap

BA E-0080

Stop Solution - Ready to use

Content:

0.25 M sulfuric acid

Volume:1 x 12 ml/vial, light grey cap

Hazards
identification:
H290 May be corrosive to metals.
BA E-2231

Kynurenine Microtiter Strips - Ready to use

Content:

1 x 96 well (12x8) antigen precoated microwell plate in a resealable pouch withdesiccant

BA E-2210

Kynurenine Antiserum - Ready to use

Content:

Rabbit anti-kynurenine antibody, blue coloured

Volume:1 x 6 ml/vial, blue cap

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Standards and Controls - Ready to useCat. no.

Component

Colour/Cap

Concentration
ng/mlConcentration
nmol/lVolume/Vial

BA E-2201

white00

4 ml

100

480

4 ml

BA E-2202

light yellow

BA E-2203

orange300

1 440

4 ml

BA E-2204

dark blue

1 000

4 800

4 ml

BA E-2205

light grey

3 000

14 4004 ml

BA E-2206

black
light green

10 00048 0004 ml

BA E-2251
BA E-2252

dark red

Refer to QC-Report for expected value andacceptable range!

4 ml
4 ml

Conversion:

Kynurenine (ng/ml) x 4.80 = Kynurenine (nmol/l)Content:

TRIS buffer with non-mercury stabilizer, spiked with defined quantity of kynurenine
Acylation Buffer - Ready to use

BA E-2211
Content:2-(N-Morpholino)ethanesulfonic acid (MES) buffer

Volume:1 x 30 ml/vial, brown cap
Acylation Reagent - Ready to use

BA E-2212
Content:acylation reagent in dimethylsulfoxide (DMSO)

Volume:1 x 3 ml/vial, green cap

4.2 Additional materials and equipment required but not provided in the kit








Calibrated precision pipettes to dispense volumes between 20 – 500 µl
Microtiter plate washing device (manual, semi-automated or automated)
ELISA reader capable of reading absorbance at 450 nm and if possible 620 – 650 nm
Temperature controlled incubator (37 °C) or similar heating device
Microtiter plate shaker (shaking amplitude 3 mm; approx. 600 rpm)
Absorbent material (paper towel)
Water (deionized, distilled, or ultra-pure)
Vortex mixer

5. Sample collection and storage
PlasmaWhole blood should be collected into centrifuge tubes containing EDTA as anti-coagulant (MonovetteTM orVacuetteTM) and centrifuged according to manufacturer’s instruction immediately after collection.
Haemolytic, icetric and lipemic samples should not be used for the assay.
Storage: up to 48 hours at 2 - 8 °C, for longer period (up to 6 month) at -20 °C.
Repeated freezing and thawing should be avoided.
Serum
Collect blood by venipuncture (MonovetteTM or VacuetteTM for serum), allow to clot, and separate serum bycentrifugation according to manufacturer’s instruction. Do not centrifuge before complete clotting hasoccurred.
Haemolytic, iceteric and lipemic samples should not be used for the assay.
Storage: up to 48 hours at 2 - 8 °C, for longer period (up to 6 month) at -20 °C.
Repeated freezing and thawing should be avoided.
6. Test procedure
Allow all reagents and samples to reach room temperature and mix thoroughly by gentle inversion before
use. Duplicate determinations are recommended. It is recommended to number the strips of the microwellplate before usage to avoid any mix-up.

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The binding of the antisera and of the enzyme conjugate and the activity of the enzyme are temperaturedependent, and the absorption values may vary if a thermostat is not used. The higher the temperature, thehigher the absorption values will be. Varying incubation times will have similar influences on the absorbance.
The optimal temperature during the Enzyme Immunoassay is between 20 – 25 °C.
In case of overflow, read the absorbance of the solution in the wells within 10 minutes, using a
microplate reader set to 405 nm.
6.1 Preparation of reagents
Wash Buffer
Dilute the 20 ml Wash Buffer Concentrate with water (deionized, distilled, or ultra-pure) to a final volume of1000 ml.
Storage: 1 month 2 – 8 °C.
Acylation Reagent
The Acylation Reagent has a freezing point of 18.5 °C. To ensure that the Acylation Reagent forms ahomogeneous, crystal-free solution when being used, it must have reached room temperature.
Kynurenine Microtiter Strips
In rare cases residues of the blocking and stabilizing reagent can be seen in the wells as small, white dots orlines. These residues do not influence the quality of the product.
6.2 Acylation
1.

Pipette 10 µl of the standards, controls and samples into the appropriate wells of the Microtiter
Plate.

2.

Add 250 µl of the Acylation Buffer to all wells.

3.

Add 25 µl of the Acylation Reagent to all wells and mix shortly.

4.

Cover the plate with Adhesive Foil and incubate 90 min at 37 °C.

5.

Use 20 µl for the ELISA!

6.3 Kynurenine ELISA
1.

Pipette 20 µl of the prepared standards, controls and samples into the appropriate wells of theKynurenine Microtiter Strips.

2.

Pipette 50 µl of the Kynurenine Antiserum into all wells and mix shortly.

3.

Cover plate with Adhesive Foil and incubate for 15 - 20 h (overnight) at 2 – 8 °C.

4.

Remove the foil. Discard or aspirate the contents of the wells. Wash the plate 4 x by adding 300 µl of
Wash Buffer, discarding the content and blotting dry each time by tapping the inverted plate onabsorbent material.

5.

Pipette 100 µl of the Enzyme Conjugate into all wells.

6.

Incubate for 30 min at RT (20 – 25 °C) on a shaker (approx. 600 rpm).

7.

Discard or aspirate the contents of the wells. Wash the plate 4 x by adding 300 µl of Wash Buffer,discarding the content and blotting dry each time by tapping the inverted plate on absorbentmaterial.

8.

Pipette 100 µl of the Substrate into all wells and incubate for 20 - 30 min at RT (20 – 25 °C) on ashaker (approx. 600 rpm). Avoid exposure to direct sunlight!

9.

Add 100 µl of the Stop Solution to each well and shake the microtiter plate to ensure a homogeneous
distribution of the solution.

10.

Read the absorbance of the solution in the wells within 10 minutes, using a microplate reader set to
450 nm (if available a reference wavelength between 620 nm and 650 nm is recommended).

7. Calculation of results
Kynurenine
63.3 – 10 000 ng/ml

Measuring range

The standard curve is obtained by plotting the absorbance readings (calculate the mean absorbance) of thestandards (linear, y-axis) against the corresponding standard concentrations (logarithmic, x-axis).
Use non-linear regression for curve fitting (e.g. spline, 4- parameter, akima).

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This assay is a competitive assay. This means: the OD-values are decreasing with increasing
concentrations of the analyte. OD-values found below the standard curve correspond to highconcentrations of the analyte in the sample and have to be reported as being positive.
The concentrations of the samples and controls can be read directly from the standard curve.
Conversion
Kynurenine (ng/ml) x 4.80 = Kynurenine (nmol/l)
Expected reference values
It is strongly recommended that each laboratory should determine its own reference value.
Plasma / Serum
237.4 – 754.2 ng/ml
7.1 Quality control
The confidence limits of the kit controls are indicated on the QC-Report.
7.2 Typical standard curve
Example, do not use for calculation!
Kynurenine
2,00
1,80
1,60
1,40

OD

1,20
1,00
0,80
0,60
0,40
0,20
0,00
10100

1000

10000

100000ng/ml
8. Assay characteristics
Analytical Sensitivity(Limit of Detection)

Kynurenine
45.7 ng/ml
Substance

Analytical Specificity(Cross Reactivity)

Cross Reactivity (%)

L-Kynurenine
5-Hydroxy-DL-Tryptophan, Tyrosin,
Phenylalanin, Serotonin,
L-Asparagin, Kynurenic Acid
Tryptophan
3-Hydroxy-DL-Kynurenin

Precision Serum
Intra-Assay
Sample1 (n = 20)
2 (n = 20)
3 (n = 20)
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100
0.05
0.25
0.36

Inter-Assay(ng/ml)
382.3
963.0
2242.0

SD
49.4
99.3
244.8

CV (%)
12.9
10.3
10.9

Effective: 2019-09-24

Sample1 (n = 20)
2 (n = 20)
3 (n = 20)(ng/ml)373.8
893.5
2050.6

SD
65
119
295

CV (%)
17.5
13.3
14.4
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Precision Plasma
Intra-Assay

Inter-Assay

SampleMean(ng/ml)SD(ng/ml)

CV (%)

SampleMean(ng/ml)SD(ng/ml)

CV (%)

1 (n = 20)
2 (n = 20)
3 (n = 20)

386.5
986.9
2383.8

57.1
90.4
278.4

14.8
9.2
11.7

1 (n = 20)
2 (n = 20)3 (n = 20)

353.7
870.1
1918.1

4362
164

12.3
7.1
8.5

Linearity

RecoverySerum
PlasmaRange Linearity %90 - 104
89 - 102

Mean Linearity %9594Serum
Sample 1
Sample 2
Sample 3
PlasmaSample 1
Sample 2
Sample 3

Range Recovery (%)
90 - 109
90 - 96
95 - 118
Range Recovery (%)
82 - 106
90 - 104
97 - 110

Mean Recovery (%)
10193109
Mean Recovery (%)
9699103

Method Comparison: ELISA
vs. LC-MS/MS

PlasmaLC-MS/MS = 0.9x + 71.5

Serial dilution up to1:128
1:128% Recovery after
spiking% Recovery afterspiking

R2 = 0.9355; N = 30

9. References/Literature(1) Lapin et al. Intensification of the central serotoninergic processes as a possible determinant of thethymoleptic effect. Lancet, 1(7586):132-6 (1969)(2) Milton et al. Relationship between interferon-gamma, indoleamine 2,3-dioxygenase, and tryptophan
catabolism. FASEB J., 5(11): 2516-22 (1991)(3) Oxenkrug et al. Tryptophan kynurenine metabolism as a common mediator of genetic and environmental
impacts in major depressive disorder: the serotonin hypothesis revisited 40 years later. Isr J Psychiatry
Relat Sci., 47(1): 56-63 (2010)(4) W.H. de Jong et al. Plasma tryptophan, kynurenine and 3-hydroxykynurenine measurement using
automated on-line solid-phase extraction HPLC-tandem mass spectrometry. J. Chromatogr., B 877: 603609 (2009)For updated literature or any other information please contact your local supplier.
Symbols:

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Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContentCaution

Catalogue
numberEffective: 2019-09-24

Contains sufficient for<n> tests

For research use
only!
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