| The OPI test is based on the principle of the highly specific immunochemical reactions between antigens and antibodies, which are used for the analysis of specific substances in urine. Major antibodies and buffers used in the OPI Test Kit are listed as follows; Goat anti-rabbit IgG (Control Line), mouse monoclonal antibody against OPI (Gold Conjugate), BSA-OPI (Test Line) and phosphate buffer and Tris buffer. The cutoff of the test is 300 ng/ml of OPI.
The OPI test cassette has a letter T and C as “Test Line” and “Control Line” in the result window. Both the “Test Line” and “Control Line” in the result window are not visible before applying any samples. The “Control Line” is used for procedural control. Control line should always appear if the test procedure is performed properly and the test reagents of the control line are working properly.
MATERIALS PROVIDED The OPI test kit contains the following items to perform the assay: 1. OPI test device. 2. Disposable sample dropper. 3. Instructions for use.
MATERIALS REOUIRED BUT NOT PROVIDED 1. Specimen collection container. 2. Clock or timer.
PRECAUTIONS 1. For professional in vitro diagnostic use only. 2. Avoid cross contamination of urine samples by using a new urine specimen container and dropper for each urine sample. 3. Urine specimens are potentially infectious. Proper handling and disposal methods should be established according to good laboratory practices. 4. Do not eat or smoke while handling specimen in the laboratory. 5. The OPI device should remain in its original sealed pouch until ready for use. 6. Do not use the test if the pouch is damaged or the seal is broken. 7. Do not use the test kit after the expiration date.
STORAGE AND STABILITY The OPI test kit should be stored at 4-30 oC in the original sealed pouch. The expiration date given was determined under normal laboratory conditions.
SPECIMEN COLLECTION AND PREPARATION 1. Fresh urine specimens do not require any special handling or pretreatment. 2. Specimens should be collected in a clean glass or plastic container. 3. If testing will not be performed immediately, specimens should be refrigerated. 4. Specimens should be brought to room temperature before testing. 5. Specimens containing precipitate may yield inconsistent test results. Such specimen must be clarified prior to assaying.
PROCEDURE OF THE TEST 1. Remove the test disk from the foil packet, and place it on a flat, dry surface. 2. Holding the sample dropper above the test disk. Squeeze 2 drops of specimen into the sample well (See following Figures) 3. Interpret the test results at 3 to 5 minutes.
INTERPRETATION OF RESULTS 1. As the test kit begins to work, a purple band will appear at the left section of the result window to show that the Control Line is working properly. This band is the Control Band. 2. The right section of the result window indicates the test results. If another purple band appears at the right section of the result window, this band is Test Band.
NEGATIVE: TWO COLOR BANDS The appearance of two purple bands within the result window indicates a negative test result. No OPI above the cut-off level has been detected. The color of the Test band may be lighter or darker than that of the Control Band.
Note. A very faint band in the right section of the result window, visible in 5 minutes, indicates that the amount of OPI in the sample is near or below the cut-off level of the test. The urine specimen should be retested, or confirmed with a more specific alternative method such as gas chromatography/mass spectrometry, before a positive determination is made.
POSITIVE: ONE COLOR BAND The appearance of only one purple band within the result window indicates the result is positive, i.e. the specimen contains OPI at a concentration above the cut-off level. The urine specimen should be confirmed with a more specific alternative method such as gas chromatography/mass spectrometry, before a positive determination is made.
INVALID: A distinct purple band should always appear in the left section of the result window. The test is invalid if no color band forms in the left section of the result window.
LIMITATIONS 1. The test is designed for use with unadulterated human urine only. 2. There is a possibility that factors such as technical or procedural errors, as well as other substances in the urine samples may interfere with the test and cause erroneous results. 3. Adulterants, such as bleach and/or alum, in urine specimens may produce erroneous results regardless of the method of analysis. If adulteration is suspected, the test should be repeated with a new sample. 4. A positive test result does not provide any indication of the level of intoxication or urinary concentration. 5. The test results read after 5 minutes may not be consistent with the original reading obtained within the 5 minutes reading period. The test must be read within 5 minutes of sample application. 6. Certain medications containing opiates or opiate derivatives may produce a positive result in any chemical and immunological assay.
EXPECTED VAI.UES The OPI test is a qualitative assay. The amount of drugs and metabolites present in the urine cannot be estimated by the assay. The assay results distinguish positive from negative samples. A positive result indicates the sample contains OPI above the cutoff concentration.
PERFORMANCE CHARACTERISTICS AMD COMPARISION STUDIES The OPI test has been shown to detect an average of 300 ng/ml or more of OPI metabolites in urine. The accuracy of the OPI was evaluated in comparison to a commercially available immunoassay. A total of 50 negative real patients urine samples (concentration of OPI range of 0-220 ng/ml) and 50 positive real patients urine samples (concentration of OPI range of 300-1035 ng/ml) were tested by both procedures. Complete agreement was observed in 100% of the samples. All positive samples and part of the negative urine sample were confirmed by GC/MS.
PRECISION AND REPRODUCIBILLITY STUDIES The precision of the OPI assay was determined by carrying out the test with serially spiked OPI urine samples. The four concentrations, of 0 ng/ml, at –25% from cutoff (225 ng/ml), at the cutoff (300 ng/ml) and +25% from the cutoff (375 ng/ml) had been tested to challenge the precision of the test device. A total of 50 tests were run at 0 ng/ml, 50 at 225 ng/ml, 200 at 300 ng/ml and 50 at 375 ng/ml. The total was 350 tests. About 99% of the samples containing drug concentrations at or more than 25% over the cutoff level consistently showed positive results.
The reproducibility studies were carried out at three different sites. The urine samples containing 0, 300 ng/ml and 900 ng/ml of OPI were tested with a total of 360 OPI test kits. The samples were tested two times in the same day, and in two different assays, each day for 20 days. This permits separate comparisons of between-day, between-assay and within-day results, which were remarkable consistent.
CUTOFF STUDIES There were a total of 200 urine samples including 50 samples containing zero, 50 samples below the cutoff (225 ng/ml), 50 samples at the cutoff (300 ng/ml) and 50 samples above the cutoff (375 ng/ml). SPECIFICITY AND INTERFERENCE STUDIES The following table lists compounds that are detected by the OPI test. The results are expressed in terms of the concentration required to produce a positive result.
Compound Conc (ng/ml) Compound Conc.(ng/ml) Codeine 300 Nalorphine 1,000 Glucuronide 300 Naloxane 100,000 Hydrocodone 1,000 Norcodeine 60,000 Hydromorphe 1,000 Oxycodone 20,000 Levophanol 5,000 Oxymorphone 50,000 Procaine 100,000 Meperidine 80,000 Thebaine 20,000
Potentially interfering chemicals such as pain medication (Acetaminophen, 20 mg/dl), protein (2000 mg/dl), glucose (2000 mg/dl), hemoglobin (500 mg/ml) and pH of 6.0, 7.0 and 8.0 were supplemented to normal urine specimens devoid of OPI. The test gave consistently negative results. The base line urine with 300 ng/ml OPI scored consistently positive.
REFERENCES 1. Urine Testing for Drugs of Abuse, National Institute for Drug Abuse (NIDA), Research Monograph 73, 1986. 2. Ambre, J. J. Anal. Toxicol. 9:241-5, 1985.
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