Instructions for use

Tryptophan ELISABA E-2700R

Tryptophan ELISA
1. Introduction
1.1 Intended use and principle of the test
Enzyme Immunoassay for the quantitative determination of Tryptophan in urine, serum and plasma
samples.
After extraction and derivatization Tryptophan 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 derivatized standards, controls and samples and the solid phase bound analytecompete for a fixed number of antibody binding sites. When the system is in equilibrium, free antigen andfree antigen-antibody complexes are removed by washing. The antibody bound to the solid phase isdetected by an 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 standard curve
prepared with known standards.
1.2 Background
L-Tryptophan is one of the essential amino acids for the human metabolism and must be part of its diet.
In humans it serves as precursor for the synthesis of the neurotransmitters serotonin and tryptamine as well
as for the synthesis of nicotinic acid and the epiphyseal hormone melatonin. Tryptophan is catabolized tokynurenine through the enzyme IDO (indoleamine-2,3-dioxygenase). Increased IDO activity is an expressionof neuro-endocrine-immunological dysregulation, which is often associated with depressive symptoms suchas bipolar disorder (manic depression). In addition Tryptophan and its metabolites regulate neurobehavioral
effects such as appetite, sleeping-waking-rhythm and pain perception.
2. Procedural cautions, guidelines, warnings and limitations
2.1 Procedural cautions, guidelines and warnings(1) This kit is intended for research use only. Users should have a thorough understanding of this protocol forthe 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) The principles of Good Laboratory Practice (GLP) have to be followed.
(3) In order to reduce exposure to potentially harmful substances, wear lab coats, disposable protective
gloves and protective glasses where necessary.
(4) 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.
(5) For dilution or reconstitution purposes, use deionized, distilled or ultra-pure water.
(6) 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.
(7) Duplicate determination of sample is highly recommended to be able to identify potential pipetting errors.
(8) 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.
(9) Incubation times do influence the results. All wells should be handled in the same order and timeintervals.
(10) To avoid cross-contamination of reagents, use new disposable pipette tips for dispensing each reagent,sample, standard and control.
(11) A standard curve must be established for each run.
(12) The controls should be included in each run and fall within established confidence limits. The confidence
limits are listed in the QC-Report.
(13) 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.
(14) 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.
(15) 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.
(16) 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.
(17) Kit reagents must be regarded as hazardous waste and disposed of according to national regulations.

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2.2 Limitations
Any inappropriate handling of samples or modification of this test might influence the results.
2.2.1 Interfering substances
Serum/Plasma
Samples containing precipitates or fibrin strands or which are haemolytic or lipemic might cause inaccurate
results.
24-hour urine
Please note the sample preparation! If the percentage of the final concentration of acid is too high, this willlead to incorrect results for the urine samples.
2.2.2 Drug interferences
There are no known substances (drugs) which ingestion interferes with the measurement of tryptophan 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 Contents of the kit
Adhesive Foil - Ready to use

BA D-0090
Contents:

Adhesive Foils in a resealable pouch

Volume:1 x 4 foils

BA D-0024
Contents:BA E-0030

Reaction Plate - Ready to use
1 x 96 well plate, empty in a resealable pouch
Wash Buffer Concentrate - Concentrated 50x

Contents:

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

Contents:

Goat anti-rabbit immunoglobulins conjugated with peroxidase

Volume:1 x 12 ml/vial, red cap

BA E-0055

Substrate - Ready to use

Contents:

Chromogenic substrate containing tetramethylbenzidine, substrate buffer and hydrogenperoxide

Volume:1 x 12 ml/black vial, black cap

BA E-0080

Stop Solution - Ready to use

Contents:

0.25 M sulfuric acid

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

Hazards
identification:
H290 May be corrosive to metals.
H314 Causes severe skin burns and eye damage.
BA E-2731

Tryptophan Microtiter Strips - Ready to use

Contents:

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

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BA E-2710

Tryptophan Antiserum - Ready to use

Contents:

Rabbit anti-tryptophan antibody, blue coloured

Volume:1 x 6 ml/vial, blue cap

Standards and Controls - Ready to use
Cat. no.

Component

BA E-2701

Colour/Cap

Concentrationµg/ml

Concentrationµmol/lVolume/Vialwhite00

BA E-2702

light yellow

2.5

12.2

4 ml

BA E-2703

orange

7.5

36.7

4 ml

BA E-2704

dark blue

25

122

4 ml

BA E-2705

light grey

75367

4 ml

BA E-2706

250

1 224

4 ml

BA E-2751

black
light green

BA E-2752

dark red

Refer to QC-Report for expected value andacceptable range!

4 ml

4 ml
4 ml

Conversion:

Tryptophan (µg/ml) x 4.89 = Tryptophan (µmol/l)Contents:Acidic buffer with non-mercury stabilizer, spiked with defined quantity of tryptophan
Assay Buffer - Ready to use

BA E-2413
Contents:

Buffer with alkaline pH

Volume:1 x 20 ml/vial, yellow cap
Equalizing Reagent - Lyophilized

BA E-2428
Contents:

Lyophilized protein

Volume:1 vial, brown cap
Q-Buffer - Ready to use

BA E-2458
Contents:

TRIS buffer

Volume:1 x 20 ml/vial, white cap

BA E-2788

PBS - Ready to use

Contents:

Phosphate Buffered Saline

Volume:1 x 20 ml/vial, orange cap

BA E-2446

D-Reagent - Ready to use

Contents:Crosslinking agent in dimethylsulfoxide

Volume:1 x 4 ml/vial, white cap

BA E-2721

Precipitating Reagent - Ready to use

Contents:

Acidic reagent for precipitation of plasma/serum proteins, red coloured

Volume:1 x 4 ml/vial, white cap

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

Calibrated precision pipettes to dispense volumes between 10 – 300 µl; 12.5 ml
Polystyrene or polypropylene tubes and suitable rack
Microtiter plate washing device (manual, semi-automated or automated)
ELISA reader capable of reading absorbance at 450 nm and if possible 620 - 650 nm
Microtiter plate shaker (shaking amplitude 3 mm; approx. 600 rpm)
Absorbent material (paper towel)
Water (deionized, distilled or ultra-pure)
Vortex mixer

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5. Sample collection and storage
PlasmaWhole blood should be collected by venipuncture into centrifuge tubes containing EDTA as anti-coagulant(Monovette™ or Vacuette™ for plasma) and centrifuged according to manufacturer’s instructions at room
temperature immediately after collection.
Fasting specimens or pre-feed specimens for children (2 - 3 hours after last meal) are advised.
Haemolytic and especially 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 (Monovette™ or Vacuette™ for serum), allow to clot, and separate serum bycentrifugation according to manufacturer’s instructions at room temperature. Do not centrifuge before
complete clotting has occurred.
Fasting specimens or pre-feed specimens for children (2 - 3 hours after last meal) are advised.
Haemolytic and especially 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.
Urine
Spontaneous urine or 24-hour urine, collected in a bottle containing 10 - 15 ml of 6 M HCl, can be used.
If 24-hour urine is used please record the total volume of the collected urine.
Storage: for longer periods (up to 6 month) at -20 °C.
Repeated freezing and thawing should be avoided. Avoid exposure to direct sunlight.
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.
The binding of the antisera and of the enzyme conjugate and the activity of the enzyme are temperaturedependent, and the absorbance values may vary if a thermostat is not used. The higher the temperature,
the higher the extinction values will be. Corresponding variations also apply to the incubation times. 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 at 2 – 8 °C
Equalizing Reagent
Reconstitute the Equalizing Reagent with 12.5 ml of Assay Buffer.
Reconstituted Equalizing Reagent which is not used immediately has to be stored in aliquots for max
1 month at -20 C and may be thawed only once.
D-Reagent
The D-Reagent has a freezing point of 18.5 °C. It must be ensured that the D-Reagent has reached room
temperature and forms a homogeneous, crystal-free solution.
Tryptophan 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 Precipitation
1.

Pipette 20 µl of the standards, controls and samples into the respective tubes.

2.

Add 200 µl PBS to all tubes.

3.

Add 25 µl Precipitating Reagent to all tubes.

4.

Mix the tubes thoroughly (vortex) and centrifuge for 15 minutes at 3,000 x g.
Take 25 µl of the clear supernatant for the derivatization.

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6.3 Derivatization
1.

Pipette 25 µl of the precipitated standards, controls and samples into the appropriate wells of theReaction Plate.

2.

Pipette 50 µl of the Equalizing Reagent into all wells.

3.

Pipette 10 µl of the D-Reagent into all wells.

4.

Cover plate with Adhesive Foil and incubate for 2 h at RT (20 – 25 °C) on a shaker (approx. 600 rpm).

5.

Pipette 100 µl of the Q-Buffer into all wells.

6.

Incubate for 10 min at RT (20 – 25 °C) on a shaker (approx. 600 rpm).

7.

Use 25 µl for the ELISA!

6.4 Tryptophan ELISA
1.

Pipette 25 µl of the prepared standards, controls and samples into the appropriate wells of theTryptophan Microtiter Strips.

2.

Pipette 50 µl of the Tryptophan 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 content of the wells. Wash the plate 3 x by adding 300 µl of
Wash Buffer, discarding the content and blotting dry each time by tapping the inverted plate on
absorbent 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 content of the wells. Wash the plate 3 x by adding 300 µl of Wash Buffer,discarding the content and blotting dry each time by tapping the inverted plate on absorbent
material.

8.

Pipette 100 µl of the Substrate into all wells and incubate for 20 - 30 min at RT (20 – 25 °C) on a
shaker (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.
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).

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7. Calculation of results
Tryptophan
1.2 - 250 µg/ml

Measuring range

The calibration 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).
This assay is a competitive assay. This means: the OD-values are decreasing with increasing concentrationsof the analyte. OD-values found below the standard curve correspond to high concentrations of the analytein 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.
The total amount of Tryptophan excreted in urine during 24 h is calculated as following:µg/24h = µg/l x l/24h
Conversion
Tryptophan (µg/ml) x 4.89 = Tryptophan (µmol/l)
Expected reference values
It is strongly recommended that each laboratory should determine its own reference values.
Plasma / Serum
9.3 – 17 µg/ml

Spontaneous urine
1.5 – 40 µg/g creatinine

7.1 Quality control
The confidence limits of the kit controls are indicated on the QC-Report.

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7.2 Typical standard curve
Example, do not use for calculation!

OD

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

Tryptophan
1.2 µg/ml
Cross Reactivity (%)

Substance

Analytical Specificity(Cross Reactivity)

Tryptophan
5-Hydroxy-L-tryptophan
Tryptamine
5-Methoxy-L-tryptophan
5-Hydroxytryptamine
5-Methoxytryptamine

Precision
Intra-Assay

100
<0.01
<0.01
<0.01
<0.01
<0.01

Inter-Assay

SampleRange (µg/ml)

CV (%)

SampleRange (µg/ml)

CV (%)

1 (n = 77)
2 (n = 78)

9.4 ± 1.0
27 ± 2.8

1111

1 (n = 16)
2 (n = 16)

9.2 ± 1.4
45 ± 415
8.4

Linearity

Recovery

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Urine

Range (µg/ml)
1.3 - 100UrineSerum

Mean (%)
10695

Effective: 2018-01-29

Serial dilution up to
1:75

Range (%)
101 - 129

Range (%)
104 - 11086 - 100

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9. References/Literature(1) El-Bakly et al. Hypericum Perforatum Decreased Hippocampus TNF-α and Corticosterone Levels with No
Effect on Kynurenine/Tryptophan Ratio in Bilateral Ovariectomized Rats. Korean J Physiol Pharmacol,
18:133-139 (2014)(2) Nowak et al. Tryptophan hydroxylase-1 regulates immune tolerance and inflammation. The Journal ofExperimental Medicine, 209(11): 2127-2135 (2012)(3) Sorensen et al. Indoleamine 2,3-dioxygenase specific, cytotoxic T cells as immune regulators. Blood,
117(7): 2200-2210 (2011)For updated literature or any other information please contact your local supplier.
Symbols:

Version: 13.0-r

Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContentCaution

Catalogue
numberEffective: 2018-01-29

Contains sufficient for<n> tests

For research use
only!

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