DRG International EIA-4161 Dopamine ELISA Instruction Manual

DRG International EIA-4161 Dopamine ELISA

Product Information

Specifications

• Product Name: Dopamine ELISA EIA-4161
• Intended Use: Enzyme Immunoassay for the quantitative determination of dopamine in plasma and urine
• Manufacturer: DRG International, Inc., USA
• Version: 19.1

Product Usage Instructions

Clinical application
Catecholamines like dopamine, adrenaline, and noradrenaline are neurotransmitters of the sympathetic nervous system in humans. They play roles in physiological processes and stress responses. The test aids in diagnosing tumors of the sympathoadrenal system and  other conditions like hypertension, cardiovascular diseases, and mental disorders.

Procedural Cautions, Guidelines, Warnings, and Limitations
warnings: This kit is for professional use only. Users must understand the protocol provided with the kit for successful testing. Strict adherence to the provided instructions is essential for reliable performance.

FAQ
Q: Can this kit be used for self-testing at home?
A: No, this kit is for professional use only and should be operated by individuals with a thorough understanding of the protocol.

Please use only the valid version of the Instructions for Use provided with the kit.

INTRODUCTION

• Intended use and principle of the test
• Enzyme Immunoassay for the quantitative determination of dopamine in plasma and urine.
• Dopamine is extracted by using a cis-diol-specific affinity gel, acylated and then converted enzymatically.
• The subsequent competitive ELISA uses the microtiter plate format. The antigen is bound to the solid phase of the microtiter plate. The acylated standards, controls and samples compete with the solid phase bound analytes for a fixed number of antibody binding sites. After the system is in equilibrium, free antigen and free antigen-antibody complexes are removed by washing. The antibody bound to the solid phase is detected by an anti-rabbit IgG-peroxidase conjugate using TMB as a substrate resulting in a colour reaction.The reaction is monitored at a wavelength of 450 nm.
• Quantification of unknown samples is achieved by comparing their absorbance with a reference curve prepared with known standard concentrations. Manual processing of the ELISA is recommended. The use of automatic laboratory equipment is the responsibility of the user. This in-vitro diagnostic is for professional use only.

Clinical application

• In humans, the catecholamines adrenaline (epinephrine), noradrenaline (norepinephrine) and dopamine are neurotransmitters of the sympathetic nervous system and are involved in many physiological processes. The sympathetic nervous system sets the body to a heightened state of  alert, also called as the body’s fight-or-flight response.
• In the human body the catecholamines and their metabolites indicate the adaptation of the body to acute and chronic stress.
• Next to the metanephrine/normetanephrine, the catecholamines are important for the diagnosis and the follow-up of tumors of the sympathoadrenal system like the pheochromocytoma. The quantitative determination of catecholamines in urine is preferred for the diagnosis of these
• tumors, whereas the determination of catecholamines in plasma is medically sensible for the localization of the tumor and for function testing. Values above the cut-off can provide an indication for neuroendocrine tumors.
• However, in literature various diseases like hypertension, cardiovascular diseases, schizophrenia and manic depression are described with abnormal low or high levels of catecholamines.
• Therapeutic consequences should never be based on laboratory results alone even if these results are assessed in accordance with the quality criteria of the method. Any laboratory result is only a part of the total clinical picture of the patient.
• Only in cases where the laboratory results are in an acceptable agreement with the overall clinical picture of the patient it can be used for therapeutic consequences.

PROCEDURAL CAUTIONS, GUIDELINES, WARNINGS AND LIMITATIONS

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 must be used to run the assay. Reliable performance will only be attained by strict and careful adherence to the instructions provided.
2. This assay was validated for a certain type of sample as indicated in Intended Use (please refer to Chapter 1). Any off-label use of this kit is in the responsibility of the user and the manufacturer cannot be held liable.
3. The principles of Good Laboratory Practice (GLP) must be followed.
4.  In order to reduce exposure to potentially harmful substances, wear lab coats, disposable protective gloves and protective glasses where necessary.
5.  If serious incidents should occur in connection with this product, they should be reported to the manufacturer and the competent national authorities.
6. All kit reagents and specimens should be brought to room temperature and mixed gently but thoroughly before use. For dilution or reconstitution purposes, use deionized, distilled, or ultra-pure water. Avoid repeated freezing and thawing of reagents and specimens.
7. The microplate contains snap-off strips. Unused wells must be stored at 2 °C – 8 °C in the sealed foil pouch with desiccant and used in the frame provided. Microtiter strips which are removed from the frame for usage should be marked accordingly to avoid any mix-up.
8. Duplicate determination of sample is highly recommended.
9. Once the test has been started, all steps should be completed without interruption. Make sure that the required reagents, materials, and devices are prepared for use at the appropriate time.
10. Incubation times do influence the results. All wells should be handled in the same order and time intervals.
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 provided with the kit.
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 case of 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 an abundant volume of water and skin with soap and abundant water. Rinse contaminated items before reuse.
17. For information about hazardous substances included in the kit please refer to Safety Data Sheet (SDS). The Safety Data Sheet for this product is made available upon request.
18. Kit reagents must be regarded as hazardous waste and disposed of according to national regulations.
19. The expected reference values reported in this test instruction are only indicative. It is recommended that each laboratory establishes its own reference intervals.
20. In case of any severe damage to the test kit or components, the manufacturer has to be informed in writing, at the latest, one week after receiving the kit. Severely damaged single components must not be used for a test run. They must be stored properly until the manufacturer decides what to do with them. If it is decided that they are no longer suitable for measurements, they must be disposed of in accordance with national regulations.
21. The results obtained with this test kit should not be taken as the sole reason for any therapeutic consequence but must be correlated to other diagnostic tests and clinical observations.

Limitations
Any inappropriate handling of samples or modification of this test might influence the results.

Interfering substances and proper handling of specimens

• Plasma
  Samples containing precipitates or fibrin strands or which are hemolytic or lipemic 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 800 mg/dl triglycerides) have no influence on the assay results.
  If the concentrations cannot be estimated and there are doubts as to whether the above limit values for hemolytic, icteric or lipemic samples are complied with, the samples should not be used in the assay.

• hour urine
  Please note the sample collection! If the percentage of the final concentration of acid is too high, this will lead to incorrect results for the urine samples.

• Drug and food interferences
  There are no known substances (drugs) which ingestion interferes with the measurement of dopamine level in the sample.

• High-Dose-Hook effect
  No hook effect was observed in this test.

STORAGE AND STABILITY

• Store the kit and reagents at 2 °C – 8 °C until expiration date.
• Do not use kit and components beyond the expiry date indicated on the kit labels.
• Once opened the reagents are stable for 2 months when stored at 2 °C – 8 °C.
• Once the resealable pouch of the ELISA plate has been opened, care should be taken to close it tightly again including the desiccant.

MATERIALS

Content of the kit

Calibration and Controls
Standards and Controls – ready to use

Component| Colour/ Cap| Concentration ng/m

| l Concentration nmol/l

| Volume/ Vial
---|---|---|---|---
| white| 0| 0| 4 ml
| yellow| 10| 65| 4 ml
| orange| 40| 261| 4 ml
| blue| 150| 980| 4 ml
| grey| 500| 3,265| 4 ml
| black| 2,000| 13,060| 4 ml
| purple| 4.5| 29| 4 ml
| green| Refer to QC report for expected value and acceptable range!| 4 ml
| red| 4 ml

Conversion
Dopamine [ng/ml] x 6.53 = dopamine [nmol/l]

Content
Acidic buffer with non-mercury stabilizer, spiked with defined quantity of dopamine for the determination of dopamine in plasma the additional Standard A/B is mandatory!

Additional materials required but not provided in the kit

• Absorbent material (paper towel)
• Water (deionized, distilled, or ultra-pure)

Additional equipments required but not provided in the kit

• Calibrated precision pipettes to dispense volumes between 10 – 700 μL; 1 mL
• 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)
• Vortex mixer

SAMPLE COLLECTION, HANDLING AND STORAGE

Plasma

• Whole blood should be collected into centrifuge tubes containing EDTA as anti-coagulant and centrifuged according to manufacturer’s instructions immediately after collection.
• In case of hemolytic, icteric or lipemic samples see 2.2.1.
• Storage: up to 6 hours at 2 °C – 8 °C, for longer period (up to 6 months) 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: up to 48 hours at 2 °C – 8 °C, up to 24 hours at room temperature, for longer periods (up to 6 months) at -20 °C. Repeated freezing and thawing should be avoided.
• Avoid exposure to direct sunlight.

TEST PROCEDURE

• Allow all reagents and samples to reach room temperature and mix thoroughly by gentle inversion before use. Number the Extraction Plate and microwell plates (Microtiter Strips which are removed from the frame for usage should be marked accordingly to avoid any mix-up). Duplicate determinations are recommended.
• The binding of the antisera and of the enzyme conjugate and the activity of the enzyme are temperature dependent. The higher the temperature, the higher 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.

The use of a microtiter plate shaker with the following specifications is mandatory: shaking amplitude 3 mm; approx. 600 rpm. Shaking with differing settings might influence the results.

Preparation of reagents and further notes

• Wash Buffer
  Dilute the 20 mL Wash Buffer Concentrate WASH-CONC 50X with water to a final volume of 1000 mL.
  Storage: 2 months at 2 °C – 8 °C

• Enzyme Solution
  Reconstitute the content of the vial ENZYME with 1 mL water (deionized, distilled, or ultra-pure) and mix thoroughly.

• Add 0.3 mL of COENZYME followed by 0.7 mL of ADJUST-BUFF.
  The total volume of the Enzyme Solution is 2.0 mL.
  The Enzyme Solution has to be prepared freshly prior to the assay (not longer than 10 – 15 minutes in advance). Discard after use!

• Dopamine Microtiter Strips
  In rare cases residues of the blocking and stabilizing reagent can be seen in the wells as small, white dots or lines. These residues do not influence the quality of the product.

• Acylation Reagent
  The ACYL-REAG has a freezing point of 18.5 °C. To ensure it is liquid when being used, it must be ensured that the Acylation Reagent has reached room temperature and forms a homogeneous, crystal-free solution before being used.

Sample preparation, extraction and acylation
*for the determination of dopamine in plasma the additional Standard A/B is mandatory!

1. Pipette 10 µL of standards, controls, urine samples and 300 µL of plasma samples into the respective wells of the EXTRACT-PLATE 48..
2. Add 250 µL of water (deionized, distilled, or ultra-pure) to the wells with standards, controls and urine samples.
3. Pipette 50 μL of ASSAY-BUFF into all wells.
4. Pipette 50 μL of EXTRACT-BUFF into all wells.
5. Cover plate with FOILS and incubate 30 min at RT (20 °C – 25 °C) on a shaker (approx. 600 rpm).
6. Remove the foil. Empty plate and blot dry by tapping the inverted plate on absorbent material.
7. Pipette 1 mL of Wash Buffer into all wells. Incubate the plate for 5 min at RT (20 °C – 25 °C) on a shaker (approx. 600 rpm). Empty plate and blot dry by tapping the inverted plate on absorbent material.
8. Pipette another 1 mL of Wash Buffer into all wells. Incubate the plate for 5 min at RT (20 °C – 25 °C) on a shaker (approx. 600 rpm). Empty plate and blot dry by tapping the inverted plate on absorbent material.
9. Pipette 150 μL of ACYL-BUFF into all wells.
10. Pipette 25 μL of ACYL-REAG into all wells.
11. Incubate 15 min at RT (20 °C – 25 °C) on a shaker (approx. 600 rpm).
12. Empty plate and blot dry by tapping the inverted plate on absorbent material.
13. Pipette 1 mL of Wash Buffer into all wells. Incubate the plate for 10 min at RT (20 °C – 25 °C) on a shaker (approx. 600 rpm). Empty plate and blot dry by tapping the inverted plate on absorbent material.
14. Pipette 175 μL of HCL into all wells.
15. Cover plate with FOILS. Incubate 10 min at RT (20 °C – 25 °C) on a shaker (approx. 600 rpm).

• Remove the foil and discard.
• Do not decant the supernatant thereafter!

The f ollowing volumes of the supernatant are needed for the subsequent ELISA:

• Dopamine (standards + urine) 25 μL
• Dopamine (plasma) 50 μL

Dopamine ELISA

1. Pipette 25 μL of the Enzyme Solution (refer to 6.1) into all wells of the Dopamine Microtiter Strips Ш DOP.
2. Pipette 25 μL of the extracted standards, controls, urine samples and 50 μL of the extracted plasma samples into the appropriate wells.
3. Add 25 μL of HCL to the standards, controls and urine samples.
4. Incubate for 30 min at RT (20 °C – 25 °C) on a shaker (approx. 600 rpm).
5. Pipette 50 μL of the DOP-AS into all wells and cover plate with FOILS.
6. Incubate for 2 h at RT (20 °C – 25 °C) on a shaker (approx. 600 rpm).
7. 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.
8. Pipette 100 μL of the CONJUGATE into all wells.
9. Incubate for 30 min at RT (20 °C – 25 °C) on a shaker (approx. 600 rpm).
10. 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.
11. Pipette 100 μL of the SUBSTRATE into all wells and incubate for 25 ± 5 min at RT (20 °C – 25 °C) on a shaker (approx. 600 rpm). Avoid exposure to direct sunlight!
12. Add 100 μL of the STOP-SOLN to all well and shake the microtiter plate shortly.
13. 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).

CALCULATION OF RESULTS

The standard curve, which can be used to determine the concentration of the unknown samples, is obtained by plotting the absorbance readings (calculate the mean absorbance) of the standards (linear, y-axis) against the corresponding standard concentrations (logarithmic, x-axis) using a concentration of 0.001 ng/ml for Standard A (this alignment is mandatory because of the logarithmic presentation of the data). Use a non-linear regression for curve fitting (e.g. 4-parameter, marquardt).
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 high concentrations of the analyte in the sample and have to be reported as being positive.

Urine samples and controls
The concentrations of the urine samples and the Controls can be read directly from the standard curve.
Calculate the 24 h excretion for each urine sample: µg/24 h = µg/L × L/24 h

Plasma samples
The read concentrations of the plasma samples have to be divided by 60.

Conversion
Dopamine [ng/mL] × 6.53 = Dopamine [nmol/L]

Expected reference value
It is strongly recommended that each laboratory should determine its own reference values.

| Dopamine
---|---
24-hour urine| < 600 µg/day

(3,900 nmol/day)

Plasma| < 100 pg/mL

Typical standard curve
Example, do not use for calculation!

CONTROL SAMPLES

It is recommended to use control samples according to national regulations. Use controls at both normal and pathological levels. Commercially obtained control samples should be treated like unknown samples. Control samples should fall within established confidence limits. The confidence limits of the kit controls are indicated on the QC Report.

ASSAY CHARACTERISTICS

Performance data

Analytical Sensitivity

| Dopamine
Limit of Blank (LOB)| Urine [ng/ml]| 2.2
Plasma [pg/ml]| 43
Limit of Detection (LOD)| Urine [ng/ml]| 2.5
Plasma [pg/ml]| 49
Limit of Quantification (LOQ)| Urine [ng/ml]| 4.8
Plasma [pg/ml]| 75
Analytical Specificity (Cross-Reactivity)

Substance| Cross Reactivity [%]
| Dopamine
Derivatized Adrenaline| 0.02
Derivatized Noradrenaline| 6.4
Derivatized Dopamine| 100
Metanephrine| < 0.01
Normetanephrine| 0.01
3-Methoxytyramine| 0.49
3-Methoxy-4-hydroxyphenyl glycol| < 0.01
Tyramine| 0.18
Phenylalanine, Caffeinic acid, L-Dopa, Homovanillic acid, Tyrosine,

3-Methoxy-4-hydroxy mandelic acid

| < 0.01
Precision

Intra-Assay Urine (n = 60)| Intra-Assay Plasma (n = 60)
| Sample| Range [ng/mL]| CV [%]| | Sample| Range [pg/mL]| CV [%]
Dopamine| 1| 82 ± 16.1| 19.7| Dopamine| 1| 75 ± 22| 29.8
2| 253 ± 41.1| 16.3| 2| 353 ± 86| 24.4
3| 714 ± 67| 9.4| 3| 1,187 ± 293| 24.9
Inter-Assay Urine (n = 33)| Inter-Assay Plasma (n = 18)
| Sample| Range [ng/mL]| CV [%]| | Sample| Range [pg/mL]| CV [%]
Dopamine| 1| 79.3 ± 18.8| 23.7| Dopamine| 1| 238 ± 67.0| 28.2
2| 222 ± 27.0| 12.1| 2| 1,072 ± 201| 18.8
3| 630 ± 69.0| 11.0| 3| 3,449 ± 491| 14.2
Recovery| | Range| Mean (%)| Range (%)
---|---|---|---|---
** Dopamine| Urine| 225 – 1,306 ng/mL| 110| 101-124
Plasma| 57.4 – 16,054 pg/mL| 89| 84-92
Linearity**

| Serial dilution up to| Mean [%]| Range [%]
Dopamine| Urine| 1:512| 104| 83 – 126
| Plasma| 1:512| 106| 85 – 132

REFERENCES/LITERATURE

1. . Kim et al. Vitamin C prevents stress-induced damage on the heart caused by the death of cardiomyocytes, through the down-regulation of the excessive production of catecholamine, TNF-α, and ROS production in GULO(-I-) Vit C-Insufficient mice. Free Radical Biology and Medicine, 65:573-583 (2013)
2. Bada et al. Peripheral vasodilatation determines cardiac output in exercising humans: insight from atrial pacing. The Journal of Physiology, 590(8):2051-2060 (2012)
3.  Parks et al. Employment and work schedule are related to telomere length in women. Occupational & Environmental Medicine 68(8):582-589 (2011)

For updated literature or any other information please contact your local supplier.

SYMBOLS USED

DRG Instruments GmbH, Germany Frauenbergstraße 18, D-35039 Marburg
Phone: +49 (0)6421-1700 0,
Fax: +49 (0)6421-1700 50
Website: www.drg-diagnostics.de
E-mail: drg@drg-diagnostics.de

References

• Analyticon® Biotechnologies GmbH
• It's Uptime | International® Trucks

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