rev:July 15, 1997
JANSSEN BIOTECH RIA KITS: SUFENTANIL RIA KIT
-note: All Janssen Biotech products are for in vitro research use only
and are not for use in or on humans or animals-Not for use in diagnostics.
These kits are only suitable for pharmacokinetic studies (not for charting
of patient results & not for drug of abuse determinations). The following
information is for illustrative purposes only. See insert that comes with
each kit for any batch specific information or recent changes in the procedure.
FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC PROCEDURES
Table of Contents
1. Introduction p 2
2. Principle of the method p 3
3. Reagents p 4
4. Equipment and materials required for the assay p 7
5. Specimen collection p 8
6. Assay procedure p 9
7. Calculation of results p 11
8. Specific performance characteristics p 14
9. Expected values p 15
10. References p 16
1 .INTRODUCTION
Sufentanil,N-[4-(methoxymethyl)-1-[2-(2-thiemyl)ethyl]-4-piperidinyl]-N-phenylpropanamide (Fig.1), is a novel,long-acting narcotic analgesic, about ten times more potent than fentanyl (1). Analgesic doses administered to surgical patients are in the range of 0.5 to 5 ug/kg (2).
For measuring sufentanil plasma levels after such low doses, a very sensitive and reliable radioimmunoassay (RIA) has been developed (3).
Depending on the sufentanil plasma concentration, the RIA can be performed in two ways.Higher-concentrated plasma samples are assayed as such but it is strongly advised to extract lower-concentrated plasma or serum samples (< 1 na/ml) to enhance the sensitivity and specificity of the assay method.
The sufentanil radioimmunoassay kit allows the rapid and sensitive determination of sufentanil plasma levels.The standard or the patient's circulating sufentanil competes with the radioactive tracer for binding sites on the sufentanil antibody. After the formation of the immunocomplex has been completed, bound and free sufentanil are separated by selective adsorption on dextran-coated charcoal. After equilibrium, the charcoal complex is removed by centrifugation and the radioactivity in the supernatant, due to specifically bound 3H-sufentanil , is determined in a liquid scintillation (B-) counter.
For more information about the general principles of radioimmunoassay, the reader is referred to some excellent textbooks(4,5,6).
This kit is intended for use in research and for diagnostic purposes only. All necessary reagents are supplied and are sufficient for at least 200 assay tubes, provided that the suggested assay protocol is followed.
3H-labelled sufentanil 1 2 vials
Standard sufentanil 2 2 vials
Sufentanil antiserum 3 2 vials
Charcoal/dextran 4 2 vials
Phosphate buffer - BSA 6 2 vials
Possible number of tubes 200
The sufentanil RIA-kit is shipped at ambient temperature and should stored
at 4'C upon receipt. Stability of the individual kit components and precautions
for handling are described below.
3.1. 3H-Sufentanil 1
Each vial contains the amount of labelled sufentanil, dissolved in methanol, sufficient for at least 100 tubes.
' Radioactivity: approx. 1.1uCi
' Storage: -methanolic solution: freeze at -20'C.
-methanol-water solution (30:70,v/v):refrigerate at 2-8'C
' Stability: refer to expiration date on vial label.
At the time of use, dilute the methanol stock solution with 4.0ml
methanol-distilled water (30:70,v/v) to obtain an appropriate solution,
corresponding to circa 25000 dpm/50 ul. The methanol-water solution cannot
be stored for longer than two weeks at 2-8'C.
WARNING
This radioactive material should only be used for in vitro laboratory tests not involving internal or external administration of the material to humans or animals.
Adherence to the basic rules of radiation safety (8) should provide adequate protection.
3.2 Sufentanil standard concentrate 2
Each vial contains sufentanil dissolved in methanol at a concentration of 2.0 ug/ml.
' Storage: refrigerate at 2-8'C.
' Stability: refer to expiration date on vial label.
At the time of use, dilute the methanolic solution with methanol according to the scheme given in chapter 6. The resulting diluted solutions cannot be stored for longer than two weeks at 2-8'C.
WARNING:
This material, dissolved in methanol, should only be used for in vitro laboratory tests not involving internal or external administration of the material to humans or animals.
The total amount of sufentanil present in the kit is approximately 4 microgram.
Each vial contains the amount of antiserum sufficient for 100 tubes. The antiserum has been raised in rabbits against a hapten derived from sufentanil. The lyophilized preparation contains bovine serum albumin and sodium azide (0.1%) as a preservative.
' Storage: refrigerate at 2-8'C
' Stability: refer to expiration date on vial label.
At the time of use, reconstitute the contents with 10 ml of distilled water. Refreezing of the diluted antiserum is not recommended. Following the procedure recommended for the kit, the antiserum binds 25-45% of labelled sufentanil.
Eash vial contains enough activated charcoal (Norit-A) and dextran (T 70) for at least 100 determinations.
' Storage: refrigerate at 2-8'C
' Stability: refer to expiration date on vial label.
Half an hour before use, suspend the preweighed dry mixture into 25 ml of distilled water to obtain a 2% dextran-coated charcoal (DCC) suspension.
For this purpose, introduce into the container a magnetic bar and put the suspension on an electromagnetic stirrer. Keep the dextran-coated charcoal in suspension while transferring into the tubes.
' Storage: refrigerate at 2-8'C.
' Stability: the suspension ca be stored for at least 7 days at 2-8'C.
Each vial contains disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium azide and bovine serum albumin.
' Storage: refrigerate at 2-8'C.
Just before use, dissolve the preweighed dry salt mixture into 50 ml of distilled water to obtain a 0.05 M buffer solution of pH 7.5, containing 2% of albumin. The minute amount of material which may cling to the vial is negligible and will not affect the pH of the solution.' Storage: refrigerate at 2-8'C
' Stability: the solution has to be used within 48 hours.
In addition to the reagents supplied with the kit, the following materials are required for the assay:
4.1. Materials required for the classic extraction
1. Blank human plasma
2. Heptane-isoamyl alcohol (98.5:1.5,v/v)
3. Sodium hydroxide, 0.5 M
4. Sulfuric acid 0.05 M
5. Ammonium hydroxide 25%
6. Glass test tubes (100 x 16 mm and 100 x 12 mm)
7. Appropriate polyethylene stoppers
8. Rotating mixer (10-15 rpm)
9. Centrifuge (3000-4000 rpm)
10. Evaporation equipment using nitrogen
4.2 Materials required for the alternative extraction
1. Materials acquired for the classic extraction (see above)
2. Extrelut 3 pre-packed glass extraction columns (Merck, Germany)
4.3 Materials required for the radioimmunoassay
1. Semi-automatic micropipettes with disposable plastic tips or an automatic sampling-dispensing system.
2. Disposable conical polypropylene tubes for microdosage (type Eppendorf 1.3 ml)
3. Vortex mixer
4. Rotating mixer (+25 rpm) or equivalent
5. Magnetic stirrer and stirring bars
6. Non-refrigerated multisample centrifuge operating at a suitable number of a (6000-8000)
7. Disposable Pasteur pipettes
8. Test tube racks
9. Liquid scintillation cocktail with a water-holding capacity for proteinaceous solutions of at least 10%.
10. Scintillation counter
11. Suitable graph paper (logit-log scale)
12. Distilled water
13. Methanol (analytical reagent grade)
Careful standardization of sampling conditions is recommended
' Anticoagulant: heparin sodium or EDTA disodium salt
' Type of sampling tube: glass of polystyrene
' Stoppers: polyethylene
Centrifuge the blood for 10 minutes and separate the plasma. The plasma samples should be frozen within a few hours after collection and stored at -20'C. Under these conditions plasma samples can be stored practically for an unlimited period.
Pack specimens to be mailed in insulated containers with dry ice.
6.1. Preparation of the standards
The 2.0 ug/ml sufentanil standard concentrate is diluted with methanol in order to obtain suitable concentrations within the range 2 - 0.02 ng/50 ul.
SUGGESTED DILUTION SCHEME:
Initial standard solution A0 100 ng/50 ul 1
0.1 ml A0 + 0.9 ml Methanol A1 10 ng/50 ul
0.2 ml A1 + 0.8 ml Methanol A2 2 ng/50 ul
0.1 ml A1 + 0.9 ml Methanol A3 1 ng/50 ul
0.3 ml A3 + 0.2 ml Methanol A4 0.6 ng/50 ul
0.4 ml A3 + 0.6 ml Methanol A5 0.4 ng/50 ul
0.5 ml A5 + 0.5 ml Methanol A6 0.2 ng/50 ul
0.5 ml A6 + 0.5 ml Methanol A7 0.1 ng/50 ul
0.4 ml A7 + 0.2 ml Methanol A8 0.06 ng/50 ul
0.5 ml A7 + 0.5 ml Methanol A9 0.04 ng/50 ul
0.5 ml A9 + -.5 ml Methanol A10 0.02 ng/50 ul
The standard curve and the clinical determinations must be run simultaneously. Careful pipetting is essential.'Label a sufficient amount of tubes for the standards and the samples; perform each assay at least in duplicate.'The sequence of the various procedure steps, as indicated in the table below for the assay of plasma or serum samples, must be respected.
'In case the plasma concentrations are out of range (0.1-4 ng/ tube), the samples can be diluted 1/10 with blank plasma, and reassayed.
'Use a dilution of phosphate buffer. BSA 6(1/4) instead of blank plasma for the assay in cerebrospinal fluid samples.
WARNING:
The dextran-charcoal mixture should be homogeneous while being transferred into the incubation tubes. For this purpose, keep stirring the suspension with an electromagnetic stirrer.
6.2.1. Direct immunoassay procedure
Pipet into Eppendorf tubes (volumes given in ml)
Step Samples Standards Zero standard NSB
1 Sample 0.05 - - -
2 Blank plasma - 0.05 0.05 0.05
3 Phosphate buffer BSA 0.5 0.50 0.50 0.50
4 Methanol 0.05 - 0.05 0.05
5 Standard dilutions - 0.05 - -
6 3H-Sufentanil 0.05 0.05 0.05 0.05
7 Mix the contents of the tubes.
8 Antiserum 3
0.10
0.10
0.10
-
9 Distilled water - - - 0.10
10 Mix the contents of the tubes and incubate for 2 hours at room
temperature under continuous rotation.
11 Dextran-charcoal 0.20
0.20
0.20
0.20
suspension 4
12 Mix the contents of the tubes and incubate for 1 hour at room
temperature under continuous rotation.
13 Centrifuge the tubes, after incubation with the DCC, at 8000 g for
5-10 min. pipet carefully the total supernatant disposable Pasteur-pipettes
or semi-automatic pipette with disposable tips) into counting vials. Mix
with a liquid scintillation cocktail (water-holding capa city for proteinaceous
solutions of at least 10%).
Determine the "total activity" of 0.05 ml 3H-sufentanil. 1 Measure the radioactivity of all tubes. The counts obtained will depend upon the efficiency of the scintillation counter used. In turn, this efficiency will actually determine the counting time. A higher counting time is required for a low- efficiency instrument.
6.3. Radioimmunoassay after extraction
In order to improve the sensitivity of the assay and to overcome problems
such as differences in the non-specific binding (NSB) of different types
of plasma, lower-concentrated samples (<1 ng/ml) should be extracted prior
to radioimmunoassay.
6.3.1. Classical extraction procedure
* Use a dilution of phosphate buffer - BSA 6 (1/4) instead of blank plasma
for the assay in cerebrospinal fluid samples
Pipet into test tubes (volumes given in ml)
Step
Samples
Standards Zero Standard
NSB
1 Sample 2.00 - - -
2 Blank plasma - 2.00 2.00 2.00
3 Methanol 0.05 - 0.05 0.05
4 Standard dilutions - 0.05 - -
5 NaOH 0.5M 1.00 1.00 1.00 1.00
6 Mix the contents of the tubes (100 x 16mm) with a vortex mixer.
7 Heptane-lsoamyl
5.00 5.00
5.00
5.00
alcohol
8 Mix the contents of the tubes with a rotary mixer for 10 min at at 10 rpm.
Centrifuge for 10 min at 4000 rpm, separate organic layer.
9 Sulfuric acid 0.05M
3.00 3.00
3.00
3.00
10 Mix the contents of the tubes with a rotary mixer for 10 min. at 10 rpm. Centrifuge for 10 min. at 2000 rpm, aspirate and discard the organic layer.
11 Ammonium hydroxide conc. 0.15
0.15
0.15 0.15
12 Mix the contents of the tubes with a vortex mixer and re-extract with
the extraction solvent.
13 Heptane-isoamyl alcohol 5.00
5.00
5.00
5.00
14 Mix the contents of the tubes with a rotary mixer for 10 min at 10 rpm.
Centrifuge for 10 min at 4000 rpm, and evaporate the organic layers combined
in the test tubes (100 x 12 mm) to dryness under nitrogen in a sample
concentrator at 60'C.
* Use a dilution of phosphate buffer -BSA 6 (1/4) instead of blank plasma for the assay in cerebrospinal spinal fluid samples.
Pipet into test tubes (volumes given in ml)
Step Samples Standards Zero Standard NSB
1. Sample dilution 2.00 - - -
2. Blank plasma - 2.00 2.00 2.00
3. Methanol 0.05 - 0.05 0.05
4. Standard dilutions - 0.05 - -
5. NaOH 0.5 M 1.00
1.00
1.00
1.00
6. Mix the contents of the tubes (100 x 12 mm) with a vortex mixer.
7. Pass through an extraction column (type Extrelut 3)
8. Heptane-isoamyl alcohol 9.00 9.00 9.00 9.00
9. Evaporate the organic layer collected in the test tubes (100 x 10 mm) to dryness under nitrogen in a sample concentrator at 60'C.
6.3.3. Radioimmunoassay procedure after extraction
The extraction residues are redissolved into 0.05ml of methanol by vigorous vortexing and further diluted by the addition of 0.50 ml of the phosphate buffer - BSA solution 6. After vortexing and centrifugation for a few minutes at 2500 rpm, the samples are finally processed following the scheme below.
* The standard curve and the clinical determinations must be run simultaneously. Careful pipetting is essential.
* The sequence of the various procedure steps, must be respected.
WARNING:
The dextran-charcoal mixture should be homogeneous while being transferred into the incubation tubes. For this purpose, keep stirring the suspension with an electromagnetic stirrer.
Pipet into Eppendorf tubes (volumes given in ml)
Step Samples Standard Zero Standard NSB
1 3H-Sufentanil 1 0 .05 0.05 0.05 0.05
2 Extraction residue 0.55 0.55 0.55 0.55
3 Mix the contents of the tubes.
4 Antiserum 3 0.10 0.10 0.10 -
5 Distilled water
-
-
- 0.10
6 Mix the contents of the tubes and incubate for 2 hours at room temperature under continuous rotation.
7 Dextran-charcoal
0.20
0.20
0.20 0.20
suspension 4
8 Mix the contents of the tubes and incubate for 1 hour at room temperature under continuous rotation.
9 Centrifuge the tubes, after incubation with the DCC, at 8000 g for 5-10 min. Pipet carefully the total supernatant (disposable Pasteur-pipettes or semi-automatic pipette with disposable tips) into counting vials. Mix with a liquid scintillation cocktail (water-holding cap acity for proteinaceous solutions of at least 10%)
Determine the "total activity" of 0.05 ml 3H-sufentanil. 1
Measure the radioactivity of all tubes. The counts obtained will depend upon the efficiency of the scintillation counter used. In turn, this efficiency will actually determine the counting time. A higher counting time is required for a low- efficiency instrument.
7.1 Description of calculation method
1. For each set of duplicates, compute the mean counts per minute(cpm).
2. Calculate the average net counts for all standards and samples by subtracting from each the average blank counts ("NSB").
3. Evaluate the binding ability of the analytical system as a percent ratio between the mean net counts for "zero standard" and "total activity".
% Bo = "zero standard" mean counts - NSB x 100
T
"total activity" mean counts - NSB
4. Express the mean net counts for each standard and unknown sample as a
percentage of the mean net counts for "zero standard".
% B = "standard or sample mean counts - NSB
x 100
B0 "zero standard" mean counts - NSB
5. Using logit-log paper, plot the normalized percent bound (% B/Bo for each
standard versus the corresponding amounts of sufentanil added in ng (see
example in Fig. 2).
6. Determine the ng sufentanil in each sample by interpolation from the standard curve.
7. Correct for sample volume and/or dilution to determine the original concentration (ng/ml) in the sample.
Note - The binding ability (Bo) of the analytical system should normally
lie within the range 25-45%.
- Any samples with concentration which are above the range of the standard
curve should be diluted with blank plasma and re-assayed. The values obtained
are then multiplied by the appropriate dilution factor.
- More detailed information about the graphical display of RIA dose-response
curves, curve=fitting and dose inter polation can be obtained from a review
by Rodbard (9).
7.2 Calculation example
| SAMPLE | Average DPM | NET DPM | % B/T | % B/Bo |
| Total Activity (T) | 25899 | - | 100 | - |
| Non specific binding NSB | 141 | - | 0.54 | - |
| Zero standard (0) | 8946 | 8845 | 34.1 | 100 |
| 0.02 ng/tube (A10) | 7732 | 7581 | 85.7 | |
| 0.04 ng/tube (A9) | 6730 | 6589 | 74.5 | |
| 0.06 ng/tube (A8) | 6067 | 5926 | 67.0 | |
| 0.10 ng/tube (A7) | 4930 | 4789 | 54.1 | |
| 0.20 ng/tube (A6) | 3351 | 3210 | 36.3 | |
| 0.40 ng/tube (A5) | 2281 | 2140 | 24.2 | |
| 0.60 ng/tube (A4) | 1671 | 1530 | 17.3 | |
| 1.00 ng/tube (A3) | 1122 | 981 | 11.1 | |
| 2.00 ng/tube (A2) | ||||
| Sample 1 | 6345 | 6204 | 70.1 | |
| Sample 2 (1/10) | 3736 | 3595 | 40.6 |
WARNING:
The data reported in the above table should only be regarded as an example and must not be employed instead of the data obtained by the user.
By interpolation on the calibration curve (Fig.2), samples 1 and 2 are found to contain 0.100 and 0.278 ng of sufentanil, respectively.
The final concentrations for sample 1 and 2 are then calculated:
sample 1: 0.100 x 20 = 2.00 ng/ml
sample 2: 0.278 x 200 = 55.6 ng/ml
8.1 Specificity
Cross-reactivity experiments demonstrated the specificity of the sufentanil antibodies by their ability to differentiate between the parent drug and other closely related compounds, including metabolites and other fentany-like analgesics (2). In view of this, it seems obvious that drugs, which may be co-medicated during anesthesia, will not interfere with the RIA determination of fentanyl. Because of sufentanil's low therapeutic plasma levels, no comparison could be made between its RIA and an alternative assay.However, there is strong evidence for the specificity of the assay when applied directly to plasma (3).
The limit of detection of the assay,l.e. the amount of sufentanil that causes at least a 10% decrease of the initial tracer binding ability, is 0.02ng contained in 1.0 ml of sample.
The extraction recovery of sufentanil from plasma was over 85% as determined in experiments with the radioactive labelled compound.
The intra-and inter-assay coefficients of variation were on average 8.4 and 13.5% over the range from 0.02 to 1 ng per test tube. Replicate analyses of control plasma samples, covering the therapeutic range of concentrations, yielded relative errors below 10% for each concentration.
9. EXPECTED VALUES
Normal sufentanil plasma levels in man, to be expected after single intravenous administration (0.5 - 5 ug/kg) can be obtain-d from a study by Bovill et al. (2).
10. REFERENCES
1. Niemegeers,C.J.E.,Schellekens,K.H.L., Van Bever,W.F.M. and Janssen,P.A.J.: Sufentanil, a very potent and extremely safe intravenous morphine-like compound in mice,rats and dogs. Arzneim-Forsch.(Drug Res.) 26, 1551-1556 (1976)
2. Bovill,J.G.,Sebel,P.S.,Blackburn,C.L. and Heykants,J.: Kinetics of alfentanil
and sufentanil: a comparison. Anesthesiology 55,, 86-93 (1983).
3. Michiels,M.,Hendriks,R. and Heykants,J.: Radioimmunoassay of the new opiate
analgesics alfentanil and sufentanil.Preliminary pharmacokinetic profile
in man. J.Pharm. Pharmacol. 35, 86-93 (1983)
4. Odell,W.D. and Daughaday,W.H., Eds. Principles of competitive protein-binding
assay. Lippincott, Philadelphia, 1971.
5. Langone,J.J. and Van Vunakis, H., Eds. Methods in Enzymology, Vol. 84,
Immunochemical Techniques, Part D, Selected Immunoassays, Academic Press,
New York,1982.
6. Kirkham,K.E. and Hunter, W.M., Eds. Radioimmunoassay Methods. Churchill
Livingstone, Edinburgh, 1971
7. National Bureau of Standards Handbook 92, "Safe Handling of Radioactive
Materials", Issued March 9, 1964. Department of Documents, U.S.-Government
Printing Office, Washington, D.C.
8. Rodbard,D. Statistical quality control and routine data processing for
radioimmunoassay and immunoradiometric assays. Clin. Chem. 20, 1255-1270
(1974)
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