Elisa Kit for Dopamine Testing in Rats, Mice and Tissue by Rocky Mountain Diagnostics
Overview and Instructions for use
Dopamine Research ELISA
1. Intended use and principle of the test
Enzyme Immunoassay for the quantitative determination of dopamine. Flexible test system for various biological sample types and volumes.
Dopamine is extracted by using a cis-diol-specific affinity gel, acylated and then converted enzymatically. The competitive ELISA kit uses the microtiter plate format. The antigen is bound to the solid phase of the microtiter plate. The derivatized standards, controls and samples and the solid phase bound analyte compete 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. The reaction is monitored at 450 nm.
Quantification of unknown samples is achieved by comparing their absorbance with a standard curve prepared with known standard concentrations.
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2. 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 the instructions 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 latex 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 the required 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 time intervals.
(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 case of 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 an abundant 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 Material Safety Data Sheet (MSDS). The Material Safety Data Sheet for this product is made available directly on the website of the manufacturer or upon request.
(17)Kit reagents must be regarded as hazardous waste and disposed according to national regulations.
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 desiccant again.
4.2 Additional materials and equipment required but not provided in the kit
− Calibrated precision pipettes to dispense volumes between 1 – 750 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
− Shaker (shaking amplitude 3 mm; approx. 600 rpm)
− Temperature controlled incubator (37 °C) or similar heating device
− Absorbent material (paper towel)
− Water (deionized, distilled, or ultra-pure)
− Vortex mixer
5. Sample collection and storage
Storage: up to 6 hours at 2 – 8 °C; for longer periods (up to 6 months) at -20 °C or –80 °C.
Advice for the preservation of the biological sample: to prevent catecholamine degradation, add EDTA (final concentration 1 mM) and sodium metabisulfite (final concentration 4 mM) to the sample.
6. Test procedure
Allow all reagents and samples to reach room temperature and mix thoroughly by gentle inversion before use. Duplicate determinations are recommended.
The binding of the antiserum and the enzyme conjugate and the activity of the enzyme are temperature dependent, and the absorbance may vary if a thermostat is not used. The higher the temperature, the higher the absorbance will be. Varying incubation times will have a similar influence 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
of 1000 ml.
Storage: 1 month at 2 – 8 °C
Reconstitute the content of the vial labelled ‘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 Adjustment Buffer. 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!
6.2 Sample preparation
The Dopamine Research ELISA is a flexible test system for various biological sample types and volumes. It is not possible to give a general advice how to prepare the samples. However, the following basics should help the researcher to fit the protocol to his specific needs.
• Avoid excess of acid: excess of acid might exceed the buffer capacity of the extraction buffer. A pH > 7.0 during the extraction is mandatory.
• Prevent dopamine degradation by adding preservatives to the sample (see Sample collection and storage).
• Avoid chaotropic chemicals like perchloric acid. The high salt content might reduce the recovery of dopamine. If your samples already contain high amounts of perchloric acid, neutralize them prior to the extraction step.
• Tissue samples can be homogenised in 0.01 N HCl in the presence of EDTA and sodium metabisulfite. Under these conditions, dopamine is positively charged which reduces binding to proteins and optimizes solubility.
• Avoid samples that contain substances with a cis-diol structure. These will reduce the recovery of the dopamine.
• It is advisable to perform a “Proof of Principle” to determine the recovery of dopamine in your samples. Prepare a stock solution of dopamine. Add small amounts (to change the native sample matrix as less as possible) of the stock solutions to the sample matrix and check the recovery.
• The used sample volume determines the sensitivity of this test. Determine the sample volume needed to determine the dopamine in your sample by testing different amounts of sample volume.
If you need any support in establishing a protocol for your specific purposes, do not hesitate to contact the manufacturer or your local distributor directly!
6.3 Extraction and acylation
The Dopamine Research ELISA offers a flexible test system for various biological sample types and volumes. Step 1 of the extraction procedure depends on the sample volume:
- in case you have sample volumes between 1 – 100 µl follow 1.1
- in case you have sample volumes between 100 – 500 µl follow 1.2
- in case you have sample volumes between 500 – 750 µl follow 1.3
Within a run it is only possible to measure samples with the same volume!
7. Calculation of results
The standard curve from which the concentrations in the samples can be read off, is obtained by plotting the absorbance readings (calculate the mean absorbance) measured for the standards (linear, y-axis) against the corresponding standard concentrations (logarithmic, x-axis).
Use a 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 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.
The concentrations of the samples taken from the standard curve have to be multiplied by a correction factor.
7.1 Quality control
The confidence limits of the kit controls are indicated on the QC-Report.
8. Assay characteristics
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