|
INTENDED USE
The Flunitrazepam Direct ELISA Kit provides only a preliminary analytical test result. A more specific alternate chemical method must be used in order to obtain a confirmed analytical result. Gas chromatography/ mass spectrometry (GS-MS) is the preferred confirmatory method. Professional judgement should be applied to any drug of abuse test result, particularly when preliminary positive results are used.
SUMMARY AND EXPLANATION
The Flunitrazepam Direct ELISA Kit is a sensitive in-vitro test to detect the presence of Flunitrazepam and 7-amino flunitrazepam in forensic samples such as whole blood, serum, plasma and urine. Flunitrazepam (Rohypnol) is available in a number of western European countries and Mexico for use as a hypnotic and anesthetic induction agent. It is administered orally or by intravenous injection in does of 2 mg. Flunitrazepam is metabolized via N-demethylation, 3-hydroxylation and glucoronidation and the reduction of the nitro group to an amine with subsequent acetylation. Over a seven day period an average of 84% of a labeled dose is eliminated in urine and 11% in feces. Major metabolites include 7-aminoflunitrazepam, 3-hydroxyflunitrazepam, 7- acetamidonorflunitrazepam and 3-hydroxy-7acetamido-flunitrazepam.
PRINCIPLE OF THE TEST
The Flunitrazepam Direct ELISA Kit is based upon the competitive binding to antibody of enzyme labeled antigen and unlabeled antigen, in proportion to their concentration in the reaction mixture. A 20 ml. aliquot of a diluted unknown specimen is incubated with a 100 ml. dilution of enzyme (Horseradish peroxidase) labeled Flunitrazepam derivative in micro-plate wells, coated with fixed amounts of high affinity purified polyclonal anti-Flunitrazepam. The wells are washed thoroughly and a chromogenic substrate added. The color produced is stopped using a dilute acid stop solution and the wells read at 450 nm. The intensity of the color developed is inversely proportional to the concentration of drug in the sample. The technique is sensitive to 0.5 ng/ml. The Flunitrazepam Direct ELISA Kit avoids extraction of urine or blood sample for measurement. It employs a Flunitrazepam directed antiserum. Due to the proprietary method of orienting the antibody on the polystyrene micro-plate much higher sensitivity is achieved compared to passive adsorption. This allows an extremely small sample size reducing matrix effects and interference with binding proteins(s) or other macromolecules.
MATERIALS PROVIDED 96 tests
1. Microwell coated plate 12x8x1
2. Positive Control 1 vial (ready to use) 2 ml
3. Negative Control 1 vial (ready to use) 2 ml
4. Enzyme conjugate: 1 bottle (ready to use) 15 ml
5. TMB Substrate: 1 bottle (ready to use) 28 ml
6. Stop Solution: 1 bottle (ready to use) 25 ml
MATERIALS NOT PROVIDED
1. Distilled or deionized water
2. Precision pipettes
3. Disposable pipette tips
4. ELISA reader capable of reading absorbance at 450nm
5. Absorbance paper or paper towel
STORAGE AND STABILITY
1. Store the kit at 2 – 8°C
2. Open the coated microplate bag only when it is at room temperature and close immediately after use.
3. The reagents are stable until expiration of the kit.
4. Do not freeze reagents.
5. Bring all reagents to room temperature.
WARNINGS AND PRECAUTIONS
1. Potential biohazardous materials:
The calibrator contain human source components, which have been tested and found nonreactive for hepatitis B surface antigen as well as HIV antibody with FDA licensed reagents. However there is no test method that can offer complete assurance that HIV, Hepatitis B virus or other infectious agents are absent. These reagents should be handled at the Biosafety Level 2, as recommended in the Centers for Disease Control/National Institutes of Health manual, "Biosafety in Microbiological and Biomedical Laboratories" 1984.
2. This kit is designed for Research Use Only.
3. The Flunitrazepam Direct ELISA Kit is to be used with human forensic samples, such as whole blood, serum, urine and plasma has not tested all possible applications of this assay. The Cutoff criteria are important in deciding the sample dilution.
4. Pipetting of samples and reagents must be done as quickly as possible and in the same sequence for each step.
5. Do not let wells dry during assay; add reagents immediately after completing the rinsing steps.
6. Do not add sodium azide to samples as preservative.
7. Do not use external controls containing sodium azide.
8. Use disposable pipet tips to avoid contaminating chromogenic substrate reagent. Discard reagent if it turns blue.
9. Do not pour chromogenic substrate back into container after use.
10. Keep reagents out of direct sunlight.
11. Do not pipette by mouth. Do not smoke, eat, or drink in the areas in which specimens or kit reagents are handled.
12. The components in this kit are intended for use as an integral unit. The components of different lots should not be mixed
13. The contact of certain reagent, above all the stopping solution and the substrate with skin, eye and mucosa has to be avoided, because possible irritations and acid burns could arise, and there exists a danger of intoxication
SPECIMEN COLLECTION AND HANDLING
1. Viscous forensic samples should always be diluted in phosphate buffered saline or distilled water prior to pippetting.
2. Urine samples should be stored at 2 – 4 0 C until use.
3. Samples should be well mixed before assay. Repeated freezing and thawing should be avoided.
4. Urine samples should be shipped refrigerated with Blue Ice or equivalent.
5. Maximum precision is required for reconstitution and dispensation of the reagents.
ASSAY PROCEDURE
All standards, samples, and controls should be run in duplicate concurrently so that all conditions of testing are the same.
1. Secure the desired number of coated strips in the frame holder.
2. Add 20 ml. of calibrators and standards to each well in duplicate.
3. Add 20 ml. of the diluted specimens in duplicate (recommended) to each well.
4. For post mortem forensic samples add 100 ml. of 100 mM Phosphate Buffer saline to each well. (Optional)
5. Add 100 ml. of the Enzyme Conjugate to each well. Tap the sides of the plate holder to ensure proper mixing.
6. Incubate for 60 minutes at room temperature (20-250 C) preferably in the dark, after addition of enzyme conjugate to the last well.
7. Wash the wells 6 times with 350 ml. distilled water using either a suitable plate washer or wash bottle taking care not to cross contaminate wells. If testing samples containing abnormally high amounts of hemoglobin (some Postmortem samples), use 10 mM Phosphate buffered saline pH 7.0-7.4. This will lower potential nonspecific binding of hemoglobin to the well, thus lowering background color.
8. Invert wells and vigorously slap dry on absorbent paper to ensure all residual moisture is removed. This step is critical to ensure that residual enzyme conjugate, does not skew results. If using an automated system, ensure that the final aspiration on the wash cycle aspirates from either side of the well.
9. Add 100 ml. of Substrate reagent to each well and tap sides of plate holder to ensure proper mixing.
10. Incubate for 30 minutes at room temperature, preferably in the dark.
11. Add 100 ml. of Stop Solution to each well, to change the blue color to yellow.
12. Measure the absorbance at a dual wavelength of 450 nm. and 650 nm. Compare average absorbance readings obtained from each unknown specimen with the average absorbance obtained from the Positive Reference Standard.
13. Wells should be read within 1 hour of yellow color development.
Example of Typical dose / response curve
The following data represent a typical dose/response curve.
7-Amino Flunitrazepam ng/ml Absorbance
0 1.755
5 0.902
10 0.620
25 0.372
The dose/response curve shown above should not be used in assay calculations. It is recommended that at least one in-house positive quality control sample be included with every assay run. A dose response curve or a cutoff calibrator should be run with every plate. If the average sample absorbance is equal to or less than the average absorbance of the laboratory positive reference standard the sample is POSITIVE for Flunitrazepam. If the average sample absorbance is greater than the average absorbance of the laboratory positive reference standard the sample is called NEGATIVE for Flunitrazepam .
Alternatively a dose response curve can be established by plotting standard concentration (abscissa) against corresponding absorbance (ordinate). Values for unknown samples are obtained by interpolation from the curve.
SPECIFIC PERFORMANCE CHARACTERISTICS
Precision
The precision of the FLUNITRAZEPAM Direct ELISA Kit has been verified by assessment of the mean, standard deviation (SD) and coefficients of variation (CV) in data resulting from repetitive assays.
Intra-assay Precision
Intra-assay precision was determined with reference controls.
A 0, 5, 10 and 25 ng/ml 7-amino Flunitrazepam standard was assayed eight times in the same assay. The results are tabulated in Table 1.
Table 1
7 amino Flunitrazepam (ng/ml) Mean Abs. S.D. C.V.%
0 1.802 0.105 5.8
5 0.884 0.127 14.4
10 0.578 0.061 10.5
25 0.358 0.036 9.8
Sensitivity
Assay sensitivity based on the minimum 7-amino flunitrazepam concentration required to produce a four standard deviation from assay Ao is 0.5 ng/ml
Specificity
The specificity of the Flunitrazepam ELISA for was determined by generating inhibition curves for each of the compounds listed below The antisera cross-reactivities are listed in Table 2.
TABLE 2
Cross-reactivities related drugs
Compound Approx. ng/ml equivalent to 10 ng 7amino-Flunitrazepam Cross-reactivities
Alprazolam >1000 <1
Alpha-OH Alprazolam >1000 <1
Bromazepam >1000 <1
Chlordiazepoxide >1000 <1
Clonazepam 500 2
Clorazepate >1000 <1
Diazepam 85 12
Flurazepam >1000 <1
Flunitrazepam 12 83
Halazepam >1000 <1
Lorazepam >1000 <1
Medazepam >1000 <1
Nitrazepam 900 1.1
Nordiazepam 800 1.25
Oxazepam >1000 <1
Prazepam >1000 < 1
Temazepam >1000 <1
Triazolam 750 1.33
Aliquots of a human urine matrix were spiked with the following compounds at a concentration of 10,000 ng/ml. None of these compounds gave values in the assay that were equal to or greater than the assay sensitivity level (0.5 ng/ml). Acetaminophen, Acetylsalicylic acid, Amphetamine, Aminopyrine, Ampicillin, Ascorbic acid, Atropine, Benzoylecgonine, Caffeine, Cocaine, Carbamazepine, Codeine, Chloroquine, Chloropromazine, Carbromal, Desipramine, Dextromethorphan, Dextropropoxyphene, 5,5- Diphenylhydantoin, 10-11-Dihydro-carbamazepine, Ethosuximide, Estriol, Estrone, Estradiol, Ethotoin, Glutethimide, Ibuprofen, Imipramine, Lidocaine, LSD, Methadone, Methadone-primary metabolite, Methaqualone, Methamphetamine, Mephenytoin, "-Methyl-"- propylsuccinimide, Methyl PEMA, Methsuximide, 4-Methylprimidone, Morphine, Meperidine, Niacinamide, Norethindrone, NNormethsuximide, Phensuximide, PEMA, Primidone, Phencyclidine, Phenothiazine, Phenylpropanolamine, Procaine, Quinine, THC-COOH
REFERENCES
1. R.C. Baselt, R.H. Cravey. Disposition of Toxic Drugs and Chemicals in Man. Chemical Toxicology Institute, Foster City 325-327. |