rev:July 15, 1997
JANSSEN BIOTECH RIA KITS: Alfentanil 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.
See also Alfentanil
technical notes:
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 14
8. Specific performance characteristics p 17
9. Expected values p 18
10. References p 19
1 .INTRODUCTION
Alfentanil, N-{1-(2-(4-ethyl-4,5-dihydr-5-oxo-1H-tetrazol-yl)ethyl)-4-(methyxymethyl)-4-piperidinyl)-N-phenylpropanamide, is a potent and very short-acting intravenous narcotic analgesic, chemically related to fentanyl(1,2).
For the measurement of alfentanil plasma levels after either a bolus injection or continuous infustion, a sensitive and reliable radioimmunoassay (RIA) has been developed (3). Depending on the alfentanil plasma concentration, the RIA-test consists of two separated procedures. Higher-concentrated plasma samples are assayed directly in diluted plasma. However, to overcome problems such as large differences in the non-specific binding of different types of plasma, the lower-concentrated plasma samples (<20 ng/ml) are extracted.
The Alfentanil radioimmunoassay kit allows the rapid and senstive determination of alfentanil plasma levels.The standard or the patient's circulating alfentanil competes with the radioactive tracer for binding sites on the alfentanil antibody. After the formation of the immunocomplex has been completed, bound and free alfentanil 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-alfentanil , 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(3,4,5,6).
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 alfentanil 1 2 vials
Standard alfentanil 2 2 vials
Alfentanil antiserum 3 2 vials
Charcoal/dextran 4 2 vials
Phosphate buffer 5 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-Alfentanil 1
Each vial contains the amount of labelled alfentanil, 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 Alfentanil standard concentrate 2
Each vial contains alfentanil 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 alfentanil. 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 alfentanil.
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, and sodium azide
' Storage: refrigerate at 2-8'C.
Just before use, dissolve the preweighed dry salt mixture into 250 ml of distilled water to obtain a 0.05 M buffer solution of pH 7.5. . 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: 1 month
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 or 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 Alfentanil standard concentrate is diluted with methanol in order to obtain suitable concentrations within the range 4- 0.1 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
0.4 ml A1 + 0.6 ml Methanol A2 4 ng/50 ul
0.5 ml A2 + 0.5 ml Methanol A3 2 ng/50 ul
0.5 ml A3 + 0.2 ml Methanol A4 1 ng/50 ul
0.3 ml A4 + 0.2 ml Methanol A5 0.6 ng/50 ul
0.4 ml A5 + 0.6 ml Methanol A6 0.4 ng/50 ul
0.5 ml A6 + 0.5 ml Methanol A7 0.2 ng/50 ul
0.5 ml A7 + 0.5 ml Methanol A8 0.1 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 in dilutions starting at 1/10 with blank plasma, and reassayed. Submit the proper dilution to the direct radiomiiunoassay procedure.
The sequence of the various procedure steps, as indicated in the table below for the assay of plasma or cerebrospinal fluid samples, must be respected.
-Be sure the standard curves are constructed accordingly as the samples, ie, using the same plasma/buffer ratios.
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 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-Alfentanil 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 overnight 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-Alfentanil. 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
Pipet into test 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 Methanol 0.05 - 0.05 0.05
4 Standard dilutions - 0.05 - -
5 NaOH 0.5M 0.05 0.05 0.05 0.05
6 Mix the contents of the tubes (100 x 16mm) with a vortex mixer.
7 Heptane-lsoamyl
3.00 3.00
3.00
3.00
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 and extract aqueous
phase again..
9 Heptane-isoamyl alcohol 2.00
2.00
2.00
2.00
10 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 the organic layers combined in the test tubes (100 X 12mm) to dryness under nitrogen in a sample concentrator at 60 DEG C.
Pipet into test tubes (volumes given in ml)
Step Samples Standards Zero Standard NSB
1. Sample dilution 0.50 - - -
2. Blank plasma - 0.05 0.50 0.50
3. Methanol 0.05 - 0.05 0.05
4. Standard dilutions - 0.05 - -
5. NaOH 0.5 M 0.05
0.50
0.50
0.50
6. Mix the contents of the tubes (100 x 12 mm) with a vortex mixer.
7. Pass through an extraction column (type Extrelut 1)
8. Heptane-isoamyl alcohol 6.00 6.00 6.00 6.00
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.
* In case the plasma concentrations are out of range (0.1-10ng/tube). the plasma samples can be diluted 1/10 with blank plasma and re-assayed.
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-Alfentanil 1 0 .05 0.05 0.05 0.05
2 Extraction residue 0.50 0.50 0.50 0.50
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 overnight 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-Alfentanil. 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
Bo "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 alfentanil 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) | 24348 | - | 100 | - |
| Non specific binding NSB | 199 | - | 0.8 | - |
| Zero standard (0) | 7020 | 6821 | 28.0 | 100 |
| 0.10 ng/tube (A8) | 5212 | 5013 | 73.5 | |
| 0.20 ng/tube (A7) | 3991 | 3792 | 55.6 | |
| 0.40 ng/tube (A6) | 2904 | 2705 | 39.7 | |
| 0.60 ng/tube (A5) | 2416 | 2217 | 32.5 | |
| 1.00 ng/tube (A4) | 1647 | 1448 | 21.2 | |
| 2.00 ng/tube (A3) | 1103 | 904 | 13.3 | |
| 4.00 ng/tube (A2) | 646 | 447 | 6.6 | |
| Sample 1 (dilution 1/10) | 3391 | 3192 | 46.8 | |
| Sample 2 (dilution 1/100) | 1031 | 832 | 12.2 |
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 0.31 and 2.04 ng of alfentanil, respectively.
The final concentrations for sample 1 and 2 are then calculated:
sample 1: 0.31 x 200 = 62.00 ng/ml
sample 2: 2.04 x 2000 = 4080 ng/ml
8.1 Specificity
Cross-reactivity experiments demonstrated the specificity of the alfentanil 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 alfentanil. The specificity was further confirmed by the perfect correlation between plasma concentrations in man measured by both gas chromatography (9) and RIA applied directly to plasma samples (3).
The limit of detection of the assay,l.e. the amount of alfentanil that causes at least a 10% decrease of the initial tracer binding ability, is 0.1ng contained in 1.0 ml of sample.
The extraction recovery of alfentanil from plasma was over 90% as determined in experiments with the radioactive labelled compound.
The intra-and inter-assay coefficients of variation were on average 4 and 5% over the range from 0.1 to 4 ng per test tube. Replicate analyses of control plasma samples, covering the therapeutic range of concentrations, yielded relative errors below 10% for both the direct RIA and the RIA after extraction.
Normal alfentanil plasma levels in man, to be expected after either a bolus injection (50 - 125 ug/kg) or continous infusion (2 - 12.5 ug/kg/h) can be obtained from several publications by Bovill (10),Camu (11(,Schuettler (12), Hug (13) and De Lange (14).
1. DeCastro,J.,Van de Water, A.,Wouters,L., Xhonneux,R.,Reneman, R. and Kay,B.: Comparative study of cardiovascular, neurological and metabolic side-effects of eight narcotics in dogs. Acta Anaesthesiol, Belg. 30,5-99 (1979)
2. Niemegeers,C.J.E. and Janssen, P.A.J.: Alfentanil, a particularly
short acting intravenous narcotic analgesic. Drug Dev.Res. 1, 83-88 (1981)
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,"Sale Handling of Radioactive
Materials". Issused 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)
9. Woestenborghs,R.,Michielsen,L. and Heykants,J.: Rapid and sensitive gas
chromatographic method for the determination of alfentanil and sufentanil
in biological samples. J. Chromatogr, 224, 122-127 (1981)
10.Bovill,J.G.,Sebel,P.S.,Blackburn,C. and Heykants,J.: The pharmacokinetics
of alfentanil (R 39209): a new opioid analgesic. Anesthesiology 57, 439-443
(1982)
11.Camu,F.,Gepts,E., Rucquoi,M. and Heykants, J.: Pharmacokinetics of alfentanil
in man. Anesth.Analg. 61, 657-661 (1982)
12.Schuettler,J. and Stoeckel,H.: Alfentanil (R 39 209) ein neues kurzwirkendes
Opioid. Pharma kokinetik und erste klinische Erfahrungen. Anaesthesist 31,
10-14 (1982)
13.Hug,C.C., De Lange,S. and Burm,A.G.L.: Algentanil pharmacokinetics in
patients before and after card iopulmonary bypass (CPB) Anesth.Analg. 62,
266 (1983)
14.De Lange,S.,De Bruijn,N. Stanley,T.H. and Boscoe,M.J.: Alfentanil-oxygen
anaesthesia: comparison of continuous infusion and frequent bolus techniques
by coronary artery surgery. Anesthesiology 55, A42 (1981)
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