Principles of a urine drug test
Much of the information below describes the principles of dip and cup tests and how to use and store them. This document applies to either Home or Workplace drug testing. You may also want to refer to instructions on how to perform a drug test.
During the test, the urine sample is allowed to migrate upward and hydrate the antibody (or drug protein)- colloidal gold conjugate. The mixture then migrates along the membrane chromatographically by the capillary action to the immobilized drug-protein (or antibody) band on the test region.
When a drug is absent in the urine, the colored antibody (or drug-protein)-colloidal gold conjugate and immobilized drug-protein (or antibody) bind specifically to form a visible line in the test region as the antibody complexes with the drug-protein.
When a drug is present in the urine, it will compete with drug-protein for the limited antibody sites. The line on the test region will become less intense with increasing drug concentration.
When a sufficient concentration of drug is present in the urine, it will fill the limited antibody binding sites. This will prevent attachment of the colored antibody (or drug-protein) – colloidal gold conjugate to the drug-protein (or antibody) on the test region.
Therefore, the presence of the line on the test region indicates a negative result for the drug and the absence of the test line on the test region indicates a positive result for the drug. A visible line generated by a different antigen/antibody reaction is also present at the control region of the test strip. This line should always appear, regardless of the presence of drugs or metabolites in the urine sample. This means that a negative urine sample will produce both test line and control line, and a positive urine sample will generate only control line.
The presence of control line serves as a built-in control, which demonstrates that the test is performed properly.
Specimen collection and handling
Most of our test kits with the exception of Saliva Oral test kits, say that they are for “Professional in-vitro diagnostic use only”. This means that they have been cleared by the FDA for use at home.
- Urine specimens may be potentially infectious.
- Proper handling and disposal methods should be established.
- Avoid cross-contamination of urine samples by using a new specimen collection container for each urine sample.
- Test device should remain sealed until ready for use.
- Do not use the test kit after the expiration date.
- A positive test result does not always mean an individual has taken the drug illegally as the drug can be administered legally.
- Do not store and or expose test kits at temperature greater than 30°C.
- Do not freeze.
Drug test overview
Preparation: If specimen, control, or test devices have been stored at refrigerated temperatures, allow them to warm to room temperature before testing.
Do not open test device seal until you are ready to perform the test.
An internal procedural control is included in the test device. A line must form in the Control band region regardless of the presence or absence of drugs or metabolites.
The presence of the line in the Control region indicates that the proper sample volume has been used and that the reagents are migrating properly.
If the line in the Control region does not form, the test is considered invalid. To ensure proper kit performance, it is recommended that the test devices be tested once a week with external controls.
External controls are available from commercial sources. It is important to make sure that the control values are within established limits. If the values of external control do not fall within established limits, the test results are invalid.
Additional controls may be tested according to guidelines or requirements of local, state, and/or federal regulations or accrediting organizations.
Limitations of the Procedure
- The assay is designed for use with human urine only.
- A positive result with any of the tests indicates only the presence of a drug/metabolite and does not indicate or measure intoxication.
- There is a possibility that technical or procedural error as well other substances as factors not listed may interfere with the test and cause false results.
- If adulteration is suspected, the test should be repeated with new sample.
Adulterant Test or Specimen Validity Test
Adulteration of urine samples may cause erroneous results in drugs of abuse test by either interfering with the drug screening test and/or destroying the drugs in the urine. Dilution of urine with water is probably the simplest urine adulteration method. Bleach, vinegar, Visine, sodium bicarbonate, sodium nitrite, Drano, soft drinks and hydrogen peroxide are the examples of compounds used to adulterate the urine sample. It is important to insure the integrity of urine samples in drugs of abuse test.
Creatinine (Cr), nitrite (Ni), pH, bleach/oxidant (Bl), and specific gravity (S.G.) are tested to determine the integrity of urine samples.
- Cr: Creatinine reacts with a creatinine indicator in an alkaline medium to forms a purplish-brown color complex. The color intensity is directly proportional to the concentration of creatinine. Urine samples with creatinine concentrations of less than 20 mg/ml are dilute, which may be indicative of adulteration.
- Ni: Nitrite reacts with the reagent’s aromatic amine to form a diazonium salt which couples with an indicator to yield a pink-red/purple color complex. Urine samples containing nitrite at levels greater than 15 mg/dl are considered adulterated.
- pH: Testing the pH value of urine sample is based on color change of indicator in different acidic or basic medium. The normal urine pH ranges from 4 to 9. Urine pH below 4 or above 9 indicates adulteration of the specimen with acids or alkalines.
- Bl: Bleach or other oxidizing agents react with an oxidant indicator to form a color complex. Observation of a blue-green, brown, or orange color indicates adulteration with bleach or other oxidizing agents.
- S.G.: Testing Specific Gravity is based on the pKa change of certain pretreated polyelectrolytes in relation to the ionic concentration. In the presence of an indicator, the colors changes from dark blue to blue-green in urine of low ionic concentration to green and yellow green in urine of higher ionic concentration. Urine specific gravity below 1.005 or above 1.025 is considered abnormal, which may indicate adulteration.
Adulterant or specimen validity test results are obtained by directly comparing the color of each test pad with the color block of Adulteration Color Comparison Chart. Potentially adulterated urine sample will produce abnormal color response. Unadulterated urine sample will produce normal color response. Example color chart shown below.