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drug conjugates and the drugs which may be present in the urinesample, for binding to antibodies. In the test procedure, a sample ofurine is placed in the Sample well of the device and is allowed to migrate upward. If the drug is present in the urine sample, itcompetes with the drug conjugate bound to the dye, for the limitedantibodies immobilized on the membrane. If the level of drug ordrug metabolite is above the cutoff level, the drug will saturate the antibodies, thus inhibiting the binding of the dye coated with drugconjugates to the antibodies on the membrane. This prevents the formation of a line on the membrane. Therefore, a drug-positiveurine sample will not generate a line at Test (T) position in theResult window, indicating a positive result from positive drug competition. A negative urine sample will generate a line at Test position in the Result window, indicating a negative result from an absence of competition with free drugs.
In addition to the Test line that may appear in the Result window,a Control line is present to confirm the viability of the test. ThisControl line (validation line) should always appear if the test isconducted properly. Polyclonal sheep anti-mouse IgG antibody is immobilized on the control line. The monoclonal antibody-dye conjugates that pass the line will be captured and produce a coloredline at the Control position (C). This works as a procedural control,confirming that proper sample volume was used and the reagent system at the Control line and the conjugate-color indicator workedproperly. If insufficient sample volume is used, there may not be a The AccuSign® AMP test is a simple, one-step, immunochromato-
Control line, indicating the test is invalid.
graphic assay for the rapid, qualitative detection of amphetamine inurine.
The AccuSign® AMP test provides only a preliminary analytical
The AccuSign® AMP test kit contains all the reagents necessary to
result. A more specific alternative chemical method must be used
in order to obtain a confirmed analytical result. Gas chromatog-
raphy, mass spectrometry (GC/MS) is the preferred confirmatory

AccuSign® AMP device. The test device contains a membrane
method. Other chemical confirmatory methods are available.
strip coated with mouse monoclonal anti-amphetamine anti- Clinical consideration and professional judgment should be
body and a pad containing drug-dye conjugate in a protein applied to any drug of abuse test result, particularly when
preliminary positive results are used.1
Amphetamines are a class of potent sympathomimetic agents withtherapeutic applications. They are chemically related to the human For in vitro diagnostic use only.
body’s natural catecholamines, epinephrine and norepinephrine.
Avoid cross contamination of urine samples by using a new Acute higher doses lead to enhanced stimulation of the central urine specimen container and dropper for each urine sample.
nervous system and induce euphoria, alertness, reduced appetite, Urine specimens are potentially infectious. Proper handling and a sense of increased energy and power.2 Cardiovascular re- and disposal methods should be established according to good sponses to amphetamine include increased blood pressure and cardiac arrhythmias. More acute responses produce anxiety, para- The AccuSign® device should remain in its original sealed
noia, hallucinations, and psychotic behavior. The effects of am- pouch until ready for use. Do not use the test if the pouch is phetamines generally last 2–4 hours following use, and the drug has a half-life of 4–24 hours in the body. About 30% of amphetaminesare excreted in the urine in unchanged form, with the remainder as Do not use the test kit after the expiration date.
hydroxylated and deaminated derivatives.3 The AccuSign® AMP test kit should be stored at 2–30°C (35–
The AccuSign® AMP test uses solid-phase chromatographic mem-
86°F) in the original sealed pouch. The expiration date was estab- brane immunoassay technology for the qualitative detection of lished under these storage conditions.
amphetamine. The test is based on the principle of the highlyspecific immunochemical reactions between antigens and antibod- ies which are used for the analysis of specific substances in Approximately 110 µL of urine sample is required for each test.
biological fluids. The test relies on the competition between the CONTROL (VALIDATION) LINE (C).
The Control/Validation line indicates:1.
If the procedure was followed properly.
If no control line appears, the test is NOT VALID.
Repeat the test using a new device, and follow the Negative () = Control line and Specific Drug line
Positive (+) = Control line only; No Specific Drug line AMP (i.e., the specimen contains AMP at a concentration above the Fresh urine specimens do not require any special handling or cutoff level). A positive test result does not provide any indication pretreatment. Specimens should be collected in a clean glass or of the level of intoxication or urinary concentration of the drug in plastic container. If testing will not be performed immediately, the sample; it only indicates the sample contains drug above the specimens should be refrigerated (2–8°C) or frozen. Specimens cutoff level in qualitative terms. should be brought to room temperature before testing.
Invalid: A distinct Control line (C) should always appear. The
Specimens containing a large amount of particulate matter may test is invalid if no Control line forms at the C position. Such tests
give inconsistent test results. Such specimens should be clarified by should be repeated with a new AccuSign® AMP test device.
centrifuging or allowing to settle before testing.
The test is designed for use with human urine only.
The test procedure consists of adding the urine sample to the There is a possibility that factors such as technical or proce- Sample well of the device and watching for the appearance of dural errors, as well as other substances in the urine sample which are not listed in Table 3 below, may interfere with the testand cause erroneous results.
Adulterants, such as bleach and/or alum, in urine specimens 1. For each test, open one AccuSign® AMP may produce erroneous results. If adulteration is suspected, the and label the AccuSign® device with the patient test should be repeated with a new sample.
The test result read after 10 minutes may not be consistent withthe original reading obtained within the 10 minute reading 2. Holding the dropper vertically, dispense 3 full period. The test must be read within 10 minutes of sample drops (110 µL) of the urine sample into the Certain medications containing amphetamines may produce a 3. Read the result after 3 minutes, but within 10 positive result in any chemical or immunological assay.
Internal Control: Each AccuSign® test device has built-in con-
trols. The Control line is an internal positive procedural control. A
Negative: The appearance of a reddish-purple Control line (C)
distinct reddish-purple Control line should always appear at the C and a line next to T indicates a negative test result; i.e., no drug position, if the test procedure is performed properly, an adequate above the cutoff level has been detected. The color intensities of the sample volume is used, the sample and reagent are wicking on the Control line and the Test line may not be equal. Any faint Test line membrane, and the test reagents at the control line and the conju- in the Result window, visible in 10 minutes, should be interpreted gate-color indicator are reactive. In addition, if the test has been as negative. A negative test result does not indicate the absence of performed correctly and the device is working properly, the back- drug in the sample; it only indicates the sample does not contain ground in the result window will become clear and provide a drug above the cutoff level in qualitative terms. distinct result. This may be considered an internal negative proce- Positive: The appearance of only a reddish-purple Control line
and no distinct line next to T indicates the test result is positive for The positive and negative procedural controls contained in each AccuSign® test device satisfy the requirements of testing a positive
control and a negative control on a daily basis. If the Control line
The precision of the AccuSign® AMP assay was determined by
does not appear at the Control position, the test is invalid and a new carrying out the test with serially diluted standard drug solutions.
test should be performed. If the problem persists, contact PBM for Ninety-five percent (95%) of the samples containing drug concen- trations 25% over the cutoff level consistently showed positiveresults.
External Control: External controls may also be used to assure
that the reagents are working properly and that the assay procedure
The study also included over 40 samples ± 25% cutoff level. These is followed correctly. It is recommended that a control be tested at results were found to be consistently in agreement with predicate regular intervals as good laboratory testing process. For informa- tion on how to obtain controls, contact PBM’s Technical Services.
Distribution of Random Error:
Twenty (20) blind samples prepared by spiking various concentra-tions of amphetamine were separately tested by two operators. The AccuSign® AMP is a qualitative assay. The amount of amphet-
test results from the two operators showed complete agreement.
amines or amphetamine metabolites present in the urine cannot beestimated by the assay. The assay results distinguish positive from negative samples. Positive results indicate the samples containamphetamines above the cutoff concentration.
The reproducibility of the test results of the AccuSign® AMP assay
was examined at three different sites using a total of 15 blind
controls, consisting of 5 negative samples, 5 moderately positivesamples (a concentration 1.5 times the cutoff level), and 5 strongly Substance Abuse and Mental Health Services Administration has positive samples (i.e., a concentration 3 times the cutoff level). The suggested that the screening cutoff for positive samples be 1000 results obtained at these three sites with these controls demon- ng/mL for amphetamine. The AccuSign® AMP test has been
strated 100% agreement with each other.
shown to detect D-amphetamine in urine at an average cutoff of1000 ng/mL.
The accuracy of AccuSign® AMP was evaluated in comparison to
The AccuSign® AMP test detects D-amphetamine and amphet-
a commercially available immunoassay (Syva® EMIT® II). A total of 480 samples was tested by both procedures. Complete agree-ment was observed in 99% of the samples as shown below (Table The following table lists compounds that are detected by the AccuSign® AMP test. The specificity of the AccuSign® AMP test
was determined by adding the drugs and drug metabolites listed to
Table 1. Accuracy: Comparison of AccuSign® AMP with
drug-negative urine specimens and testing with the AccuSign®
Syva® EMIT® II
AMP test kit. The results are expressed in terms of the concentra-
tion required to produce a positive result (Table 3).
Table 3. Specificity
In a separate study, AccuSign® AMP was evaluated against
specimens confirmed as positive by GC/MS. Of 56 samples confirmed as positive, 55 samples were positive when tested with
AccuSign® (98% agreement, Table 2).
The following compounds show no cross-reactivity when tested Table 2. Accuracy: Comparison of AccuSign® AMP
with AccuSign® AMP at a concentration of 100 µg/mL (Table 4).
with GC/MS Assay
Table 4. Non Cross-Reacting Compounds
Hawks RL, Chiang CN, eds. Urine Testing for Drugs of Abuse.
Rockville, MD: National Institute for Drug Abuse (NIDA), Research Baselt RC. Disposition of Toxic Drugs and Chemicals in Man. 2nd Ed., Davis, CA: Biomedical Publ.; 1982.
Blum K. Handbook of Abusable Drugs. 1st Ed., New York, NY: In Vitro Diagnostic Medical Device “Use By” date in year-month-day format AccuSign® is a Registered Trademark of Princeton BioMeditech Corporation.
MT Promedt Consulting GmbH
Princeton BioMeditech Corporation



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