多巴胺英文说明书 DA-北京华英生物技术研究所

Instructions for use

Dopamine RIA

¹²⁵I – Radioimmunoassay for the quantitative determination of Dopamine in plasma and urine

KIPL 0300

1. Principle of the test

Dopamine is extracted by using a cis-diol-specific affinity gel, acylated and then derivatized enzymatically. The assay procedure follows the basic principle of radioimmunoassay, involving competition between a radioactive and a non-radioactive antigen for a fixed number of antibody binding sites. The amount of 125I-labelled antigen bound to the antibody is inversely proportional to the analyte concentration of the sample. When the system is in equilibrium, the antibody bound radioactivity is precipitated with a second antibody in the presence of polyethylene glycol. The precipitate is counted in a gamma counter. Quantification of unknown samples is achieved by comparing their activity with a reference curve prepared with known standards.

2. Storage and stability

The reagents should be stored at 2 -8 �C until expiration date. Do not use components beyond the expiry date shown on the kit labels.

3. Contents of the kit

BA 0310	Dopamine Antiserum 1 x 2.75 mL	from rabbit, ready for use, green coloured, green screw cap
BA 0320	125I – Dopamine 1 x 3 mL	activity < 100 kBq, ready for use, red coloured, green screw cap
BA 1601	Standard A 1 x 1 mL	ready for use
BA 1609	Standard A/B* 1 x 1 mL	ready for use
BA 1602	Standard B 1 x 1 mL	ready for use
BA 1603	Standard C 1 x 1 mL	ready for use
BA 1604	Standard D 1 x 1 mL	ready for use
BA 1605	Standard E 1 x 1 mL	ready for use
BA 1606	Standard F 1 x 1 mL	ready for use
BA 1611	Acylation Buffer 1 x 20 mL	ready for use
BA 1612	Acylation Reagent 1 x 1.5mL	ready for use
BA 1613	Assay Buffer 1 x 4 mL	ready for use, contains 1 M HCl
BA 1614	Coenzyme 1 x 0.75 mL	ready for use, S-adenosyl-L-methionine
BA 1615	Enzyme 2 x 1 mL	lyophilized, contains the enzyme COMT
BA 1617	Extraction Buffer 1 x 4 mL	ready for use
BA 1618	Extraction Plate 1 x 48 wells	coated with boronate affinity gel
BA 1619	Hydrochloric Acid 1 x 20 mL	ready for use, yellow coloured, contains 0.025 M HCl
BA 1651	Control 1 1 x 1 mL	ready for use
BA 1652	Control 2 1 x 1 mL	ready for use
BA 3030	Precipitating Reagent 1 x 55 mL	ready for use, goat anti-rabbit serum in PEG phosphate buffer Mix thoroughly before use!
BA 3050	Adjustment Buffer 1 x 4 mL	ready for use

*For the determination of Dopamine in plasma the additional Standard A/B is mandatory!

4. Additional materials and equipment required but not provided with the kit

-Calibrated variable precision micropipettes (e.g. 10-100 µL / 100-1000 µL) -Polystyrene tubes and suitable rack

-Temperature controlled water bath, heating block or incubator (37 �C)

-Centrifuge capable of at least 3,000 x g

-Suitable device for aspirating or decanting
-Shaker (shaking amplitude 3mm; approx. 600 rpm)

-Gamma counter

-Vortex mixer

-Absorbent material (paper towel)

-Distilled water

5. Sample collection and storage

Repeated freezing and thawing of the samples should be avoided.

Plasma

EDTA-Plasma should be used. Do not use haemolytic or lipemic samples.
Storage: up to 6 hours at 2 -8°C, for longer period (up to 6 months) at -20°C.

Urine

Spontaneous urine or 24-hour urine, collected in a bottle containing 10-15 mL of 6 M HCl.
Storage: for longer period (up to 6 month) at -20°C. Avoid exposure to direct sunlight.

6. Test procedure

Allow all reagents – with the exception of Precipitating Reagent -to reach room temperature and mix thoroughly by gentle inversion before use. Number the assay tubes accordingly. Duplicate determinations are recommended.

Pipetted liquids should not adhere to the wall of the RIA tubes. If necessary please centrifuge the

tubes for 1 minute at 500xg to spin down adhering liquids.

6.1 Preparation of reagents

Enzyme solution

Reconstitute the content of the vial labelled ‘Enzyme’ with 1 mL distilled water 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, extraction and acylation

6.3 Dopamine RIA

2 Calculation of results

*For the determination of Dopamine in plasma the additional Standard A/B is mandatory!

1.	Pipette 15 µL of standards, 15 µL of controls, 15 µL of urine samples and 600 µL of plasma samples into the respective wells of the Extraction Plate.
2.	Add 500 µL of distilled water to the wells with standards, controls and urine samples.
3.	Pipette 50 µL of Assay Buffer into all wells
4.	Pipette 50 µL of Extraction Buffer into all wells
5.	Cover plate with adhesive foil and incubate 30 min at RT (20-25°C) on a shaker (approx. 600 rpm).
6.	Remove the foil and discard. Empty plate and blot dry by tapping the inverted plate on absorbent material.
7.	Pipette 1 mL distilled water into all wells. Incubate the plate for 5 min at RT (20-25°C) on a shaker (approx. 600 rpm). Empty plate and blot dry by tapping the inverted plate on absorbent material.
8.	Pipette 150 µL of Acylation Buffer into all wells.
9.	Pipette 25 µL of Acylation Reagent into all wells.
10.	Incubate 15 min at RT (20-25°C) on a shaker (approx. 600 rpm).
11.	Empty plate and blot dry by tapping the inverted plate on absorbent material.
12.	Pipette 1 mL distilled water into all wells. Incubate the plate for 5 min at RT (20-25°C) on a shaker (approx. 600 rpm). Empty plate and blot dry by tapping the inverted plate on absorbent material.
13.	Pipette 200 µL of Hydrochloric Acid into all wells.
14.	Cover plate with adhesive foil. Incubate 10 min at RT (20-25°C) on a shaker (approx. 600 rpm). Remove the foil and discard. *Do not decant the supernatant thereafter!* The following volumes of the supernatant are needed for the RIA: Dopamine 75 µL

1.	Pipette 75 µL of Hydrochloric Acid into the tubes for the NSB.
2.	Pipette 75 µL of the extracted standards, controls and samples into the respective tubes.
3.	Pipette 25 µL of Enzyme Solution (refer to 6.1) into all tubes (except totals).
4.	Mix thoroughly and incubate for 30 minutes at 37 °C.
5.	Pipette 50 µL of the 125I Dopamine into all tubes.
6.	Pipette 50 µL of Dopamine Antiserum into all tubes (*except totals and NSB*); mix thoroughly.
7.	Cover tubes. Incubate for 15 -20 hours (overnight) at 2-8°C. *Alternatively* incubate for 2 hours* at RT (20-25°C) on a shaker (approx. 600 rpm).*
8.	Mix the chilled (2 -8 °C) Precipitating Reagent thoroughly, pipette each 1 mL into all tubes (*except totals*), and mix on a vortex.
9.	Incubate for 15 minutes at 2 -8 °C.
10.	Centrifuge for 15 minutes at 3,000 x g, if possible in a refrigerated centrifuge.
11.	Decant or aspirate the supernatant carefully (*except totals*). Blot the tubes dry and leave them upside for 2 minutes.
12.	Count all tubes for 1 minute in a gamma counter.

	Concentration of the standards
Standard	A	B	C	D	E	F	A/B
Dopamine (ng/mL)	0	10	40	160	640	2,560	2.5
Dopamine (nmol/L)	0	65.3	261	1,045	4,179	16,717	16.33
Conversion:	Dopamine (ng/mL) x 6.53 = Dopamine (nmol/L)

Subtract the mean cpm of the non-specific binding NSB from the mean cpm of standards, controls and
samples.
The calibration curve from which the concentrations of the samples can be read off, is obtained by
plotting the percentage of (B-NSB)/(B0-NSB) 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).

Urine samples and controls:
The concentrations of the urine samples and the Controls 1 & 2 can be read directly from the standard
curve.

Plasma samples:
The read concentrations of the plasma samples have to be divided by 40.

7.1 Quality control

It is recommended to use control samples according to state and federal regulations. Use controls at both normal and pathological levels. The kit or other commercial controls should fall within established confidence limits. The confidence limits of the kit controls are printed on the QC-report.

7.2 Typical calibration curve (Example, do not use for calculation!)

%B/Bo

1 90 80 70 60 50 40 30 20 10 0

0,1 1 10 100 1 10000 ng/ml

8. Assay characteristics

Expected Reference Values

Dopamine

Urine

< 600 µg/day (3,900 nmol/day)

Plasma

< 100 pg/mL

Analytical Sensitivity (Limit of Detection)

Mean signal (Zero-Standard) -2SD

Dopamine

Urine

0.8 ng/mL

Plasma

20 pg/mL

Analytical Specificity (Cross Reactivity)

Substance

Cross Reactivity (%)

Dopamine

Derivatized Dopamine

100

Derivatized Adrenaline

0.03

Derivatized Noradrenaline

0.87

Metanephrine

<0.007

Normetanephrine

0.008

3-Methoxytyramine

0.55

3-Methoxy-4-hydroxyphenylglycol

<0.007

Tyramine

0.13

Phenylalanine

< 0.007

Caffeinic acid

< 0.007

Homovanillic acid

< 0.007

Tyrosine

< 0.007

3-Methoxy-4-hydroxymandelic acid

< 0.007

L-Dopa

< 0.007

Precision

Intra-Assay

Inter-Assay

Sample

Range (ng/mL)

CV (%)

Sample

Range (ng/mL)

CV (%)

Dopamine

72.94 ± 5.94

8.1

Dopamine

107.22 ± 9.66

9.0

260.1 ± 15.8

6.1

506.66 ± 67.84

13.4

Linearity

Range

Serial dilution up to

Range (%)

Dopamine

Urine

42 -966 ng/mL

1:16

89 – 113

Plasma

2,000-28,700 pg/mL

1:16

82 – 119

Recovery

Mean (%)

Range (%)

% Recovery after spiking

Dopamine

Urine

109

96 – 127

Plasma

61 – 100

Method Comparison versus HPLC*

Dopamine

Urine

HPLC = 1.07 RIA + 0.01

r = 0.98; n = 21

Plasma

HPLC = 1.00 RIA + 0.003

r = 0.96; n = 20

*The concentrations were assessed using both the RIA and the HPLC method (external QC samples from UK NEQAS). The correlation between RIA and HPLC is excellent. Please take in mind, that the UK control values are the mean of about 40 different HPLC users, and contain always one pathological sample per sending.

9. Advice on handling the test

9.1 Reliability of the test results

In order to assure a reliable evaluation of the test results it must be conducted according to the instructions included and in accordance with current rules and guidelines (GLP, RILIBÄK, etc.). Special attention must be paid to control checks for precision and correctness during the test; the results of these control checks have to be within the norm range. In case of significant discrepancies between the pre-set assay characteristics of this test and the actual results please contact the manufacturer of the test kit for further instructions. It is recommended that each laboratory establishes its own reference intervals. The values reported in this test instruction are only indicative. The results obtained with this test kit should not be taken as the sole reason for any therapeutic consequence but have to be correlated to other diagnostic tests and clinical observations.

9.2 Complaints In case of complaints please submit to the manufacturer a written report containing all data as to how the test was conducted, the results received and a copy of the original test printout. Please contact the manufacturer to obtain a complaint form and return it completely filled in to the manufacturer.

9.3 Warranty This test kit was produced according to the latest developments in technology and subjected to stringent internal and external quality control checks. Any alteration of the test kit or the test procedure as well as the usage of reagents from different charges may have a negative influence on the test results and are therefore not covered by warranty. The manufacturer is not liable for damages incurred in transit.

9.4 Disposal Residual substances and/or all remaining chemicals, reagents and ready for use solutions, are special refuse. The disposal is subject to the laws and regulations of the federation and the countries. About the removal of special refuse the responsible authorities or refuse disposal enterprises inform. The disposal of the kit must be made according to the national official regulations. Legal basis for the disposal of special refuse is the cycle economic-and waste law. The appropriate safety data sheets of the individual products are available on the homepage. The safety data sheets correspond to the standard: ISO 11014-1.

9.5 Interference Do not mix reagents and solutions from different lots. Consider different transport and storage conditions. Inappropriate handling of test samples or deviations from the test regulation can the results affect. Use no kit components beyond the expiration date. Avoid microbiological contamination of the reagents and the washing water. Consider incubation periods and wash references.

9.6 Precautions

Observe the incubation periods and washing instructions. Never pipette by mouth and avoid contact of reagents and specimens with skin. No smoking, eating or drinking in areas where samples or kit test tubes are handled. When working with kit components or samples, always wear protective gloves and wash your hand thoroughly as soon as you have finished the work. Avoid spraying of any kind. Avoid any skin contact with reagents. Use protective clothing and disposable gloves. All steps have to be performed according to the protocol. Optimal test results are only obtained when using calibrated pipettes. Sodium azide could react with lead and copper tubes and may form highly explosive metal azide. When clearing up, rinse thoroughly with large volumes of water to prevent such formation. The radioactive material may be received, acquired, possessed, and used only by physicians, veterinarians in the practice of veterinary medicine, clinical laboratories or hospitals and only for in vitro clinical or laboratory tests not involving internal or external administration of the material, or the radiation there from, to human beings or animals. Its receipt, acquisition, possession, use, and transfer are subject to the regulations and a general license of the U.S. Nuclear Regulatory Commission or of a State with which the Commission has entered into an agreement for the exercise of regulatory authority. In no case the product must be administered to humans or animals. This kit contains ¹²⁵Iodine (half life: 60 days), emitting ionizing X-(28 kev) and G-(35.5 kev) radiations. All radioactive handling should be executed in a designated area, away from regular passage. A log book for receipt and storage of radioactive materials must be kept in the lab. Laboratory equipment and glassware, which could be contaminated with radioactive substances, should be segregated to prevent cross contamination of different radioisotopes. Any radioactive spills must be cleaned immediately in accordance with the radio safety procedures. The radioactive waste must be disposed of following the local regulations and guidelines of the authorities holding jurisdiction over the laboratory. Adherence to the basic rules of radiation safety provides adequate protection. All reagents of this test kit which contain human or animal serum or plasma have been tested and confirmed negative for HIV I/II, HbsAg and HCV by FDA approved procedures. All reagents, however, should be treated as potential biohazards in use and for disposal.

Information about clinical significance or other information about the test are available through our technical support department, tech.support@biosource.be.

Symbols:

Contains sufficient for tests	Batch code	Expiry date
Catalogue number	Content	Consult instructions for use
For in-vitro diagnostic use only!	Caution
Manufacturer	Storage temperature