Confirmatory tests for Urine
The confirmatory tests for urine are based on the detection of specific biomarkers that are unique to urine and can be used to confirm if the origin of the suspected stain is human urine. The most commonly used confirmatory test for urine is the RSID-Urine test, described in detail below:
RSID-Urine test
The Rapid Stain Identification test for urine or RSID-Urine test is a lateral flow immunochromatographic strip test designed to detect the presence of Tamm-Horsfall protein (THP). THP, also known as uromodulin, is a glycoprotein that is exclusively produced by the kidney and excreted in the urine, making it a reliable marker for the detection of urine. This test can detect as little as 0.5 μL of human urine and is designed for fast, easy, and reliable detection from a variety of samples encountered at the crime scene.
Principle of the RSID-Urine test:
Figure: Principle of RSID test for the detection of Urine
Immunochromatographic assays (including the RSID-Urine test) are based on the principles of capillary action and specific antigen-antibody interactions to enable rapid and visual detection of target analytes.
This test utilizes a test strip containing distinct zones, including a sample application pad, a conjugate pad containing labelled primary antibodies (specific to THP), a reaction or test zone (T) with immobilized primary antibodies (specific to THP), and a control zone (C) with immobilized secondary antibodies (specific to primary antibody).
When a sample is added to the application pad, it begins to migrate along the membrane facilitated by capillary action. If the target antigen (THP) is present in the test sample, it forms an antigen-antibody complex with labelled primary antibodies on the conjugate pad. These complexes are captured at the test zone (T) and the control zone (Z), resulting in the formation of coloured lines at these zones. The appearance or absence of these lines at the test zone (T) indicates the test result. Since the control zone contains immobilized secondary antibodies specific to primary antibodies, a coloured line will be formed at the control zone (C) even if the target antigen is absent in the test sample and the antigen-antibody complex is not formed. This ensures proper flow and functionality of the test.
📌 Read: Immunochromatographic Assay
Procedure:
Combine the suspected urine stain extract (maximum 20 μL) with RSID-Universal Buffer to bring the test sample to a total volume of 100 μL.
Add the test sample to the sample window and keep it undisturbed for 10 minutes.
Observation:
a) A visible red line at the Control (C) position only, indicates a negative result, i.e. THP could not be detected.
b) Visible red lines at both the Control (C) and Test (T) positions indicate a positive result and confirm the presence of urine, i.e. THP is detected.
c) A visible red line at the Test (T) position only indicates an inconclusive or failed test.
Figure: Results for the RSID-Urine test