Q: What is a ‘shadow band’ and how do I interpret this result?

We define a ‘shadow band’, as a hazy, barely visible link (so faint that the color of the line is indistinct) that appears at the test line near the 10 minute time window. Often there is disagreement between laboratory members as to whether a band is present at all – another indication that this is shadow band. Shadow bands should be interpreted as a negative result

We hypothesize that these "shadow bands" are due to non-specific binding at the test line from proteins or other molecules present in the sample. The antibody striped at the test line is a charged molecule (contains positive and negative charges throughout its protein sequence) and although it specifically binds the targeted antigen due to its tertiary structure, in rare cases, components from a complex sample can bind through charge-charge interactions causing a faint accumulation of labeled antibody at the test line. The appearance of shadow bands can also increase if the flowrate across the strip is decreased due to a viscous sample; because the sample crosses the test line at a slower rate this allows more time for charge-charge interactions to occur.

We have optimized components in the buffers to minimize this effect, so shadow bands are rare – using non-approved buffers will increase the chance of shadow bands appearing – please use the provided buffers for the best results.

Q: How do I interpret a band that appears at the test line after the 10 minute test window?

Only test lines that appear within the 10 minute test window are considered positive. The RSID cassette should NOT be evaluated after 10 minutes because non-specific interactions can develop over time, resulting in the appearance of a band at the test line that is not specific to the analyte.

Q: Why does a band sometimes appear at the test line after the ten minute test window?

Some background to how lateral flow tests are constructed and how they work is required to best explain the occurrence of non-specific bands after the ten minute test window.

Lateral flow strip tests use a dual antibody approach in which a specific antigen is “sandwiched” between two antibodies that each recognize a distinct epitope on the antigen. The detection antibody is conjugated to colloidal gold through positively charged amino acid residues of the antibody binding to the negatively charged colloidal gold. A blocking reagent is used (in this case BSA) to bind to the negative charges on the colloidal gold not bound by the antibody. The binding of the blocking reagent to the unbound negative charges on the colloidal gold acts to “block” the sites from nonspecific interactions that may occur with charged molecules encountered in a forensic sample. The second antibody (the capture antibody) is striped onto the test line region of the membrane and it “captures” the antigen/antibody-gold conjugate as it moves across the test line if antigen is present in the sample.

The two main mechanisms that can contribute to bands appearing after the ten minute test window:

1. Nonspecific positive/negative charge interactions that accumulate over time

2. Backflow of sample after the completion of the test

Antibodies striped at the control and test lines of the membrane are inherently charged molecules, with positive and negative charges throughout their protein structure. Antibodies bind specifically to the antigen through its tertiary, or 3D, structure, however, components in forensic samples can bind to the test line through charge-charge interactions. We have optimized components in the buffers to minimize the effect of non-specific charge-charge interactions, so only specific interactions should occur within the ten minute test window.

Background in the lateral flow derives from non-specific binding of the detection antibody (which is conjugated to colloidal gold) to the test line – under normal circumstances this should only happen when there is a ‘bridge’ of antigen between the immobilized capture antibody and the mobile detection antibody; No antigen, no bridge, no signal – this is the theory that underlies all of these types of tests. However, over the time of the test, backflow of sample can occur from the wick at the top of strip test in the reverse direction towards the sample well. This “backflow” can allow non-specific interactions to occur from components in the sample as the fluid flows in the opposite direction under (now) completely different conditions than were present when the test was first run.

Q: Are the RSID tests qualitative or quantitative? In other words, does the intensity of the test line indicate how much antigen is present in the sample?

All four RSID tests are qualitative. In other words, a positive test line means “yes” there is antigen present and the absence of a test line means that “no” there is not antigen present or it is below the limit of detection. The lateral flow tests are not calibrated for quantification, therefore, estimating the amount of antigen present based on the intensity of the test line is not possible.

Q: Is it true that I can extract samples to test for semen or saliva for only 10 seconds?

Yes, we have found that samples can be extracted for as little as 10 seconds when testing for semen or saliva.

A series of time course experiments were undertaken with control swabs, aged samples (several years old), trace semen and saliva samples, and semen or saliva on fabrics. These data clearly demonstrated that similar results could be obtained from all tested sample types using incubation times as short as 10 seconds to as long as 1 hour (to view the data, go to www.ifi-test.com/rsidtm-documentation). Room temperature extraction of forensic samples for a minimum of 10 seconds is sufficient for detection of semenogelin with RSID™-Semen or α-amylase with RSID-Saliva. Longer incubation times (i.e., 5-60 minutes) are optional.

Please note that RSID-Blood and RSID-Urine require the longer extraction times, at least 20 minute but preferably up to one hour, especially with older samples. The target antigens associated with RSID-Blood and RSID-Urine, Glycophorin A and Tamm-Horsfall, respectively, are more difficult to extract, and thus require longer extraction times, as compared to semenogelin and α-amylase.

Q: Can I use an RSID cassette and/or RSID buffer after the expiration date?

We do not recommend using an RSID cassette or RSID buffer after the printed expiration date. Due to our QA/QC procedures, we are only able to support results obtained when the RSID cassettes or RSID buffers are used before the expiration dates.

Q: How do I interpret a weak control line?

The control line on the lateral flow tests is designed to indicate that the test has run correctly and the components of the strip test are working properly. In most cases, the control line is dark and clearly positive. However, on rare occasions, the control line may be weaker than expected. The cause of this is not always clear, although a viscous sample may cause this effect. As long as the control line is clearly positive, albeit weak, the results from the test line can be properly and accurately interpreted.

Q: What do I do if I suspect a high dose Hook effect?

A high dose Hook effect refers to the reduction or decrease of the test line signal on immunochromatographic strip tests when very high levels of target are present in the tested sample. Under these conditions, unbound target antigen can reach the test line before the colloidal gold-labeled antibody-bound antigen, thereby occupying the test line antibody sites and possibly resulting in a weaker test line signal; in extreme cases the test line signal can be reduced such that a false negative result could be observed.

If a high dose hook effect is suspected, dilute your sample 1:10 in RSID running or extraction buffer and rerun the sample and observe the result. For example, if 20 µL of extract was previously loaded onto the cassette, dilute the original sample 1:10 (10 µL of this same extract added to 90 µL of buffer in a separate tube). Add 20 µL of this diluted extract / sample to 80 µL of RSID buffer and load the 100 µL of the diluted extract + buffer onto the cassette. If the test line intensity decreases (as compared to the result from the undiluted sample), the original weak or negative result was due to low (or absent) amount of antigen. If the test line intensity increases, the original result was due high dose hook effect and your sample is positive for the antigen.

Q: How do I interpret a gray or non-red spot that may appear on the test near the test line?

Any visible non-red or gray spot or mark on the membrane should not be interpreted as positive even it is near the test line. Only a complete, side to side, red line at the test position should be interpreted as a positive result. Non red spots on the membrane after the completion of a test are rare, and are most likely due to the presence of a contaminant in a complex sample that has accumulated in a nick or scratch of the membrane. As part of our QA/QC procedures, the various lateral flow components are examined to assess the presence of flaws or nicks in the membrane before the strips are assembled. Any strips found to have nicks or flaws in the membrane are discarded during production -occasionally nicks or scratches too small to observe can be present and can be revealed after the test is run.

Q: Can I use filter paper as a substrate for my positive control?

The RSID tests are designed to be used with typical forensic evidence - this means swabs or stains on fabric. From swabs or stains on fabric an extract or soak is made in the provided buffer. By using swabs as the primary evidence, the cotton batting (the wound up threads) acts as a filter and 'cleans up' the body fluid so that the large biomolecules are retained - in very real terms the fabric/swabs act like a filter.

When body fluids are placed on filter paper, there is much less filter and this can negatively affect the RSID tests - that is why we specify that positive controls need to be made on swabs or fabric - this is better for the tests, better because is mimics actual forensic evidence and makes the tests more reliable - there are dozens and dozens of different kinds of filter paper.

If you use a very small stain on the filter paper you can probably 'sneak by' as the volume of body fluid is small and the interference with the RSID test will also be small - larger stains on filter paper will present a problem. This is similar to the issues of using body fluids directly in the RSID tests - you may recall that some labs like to add blood, saliva, etc., directly to the RSID cassette and that is a bad idea - same issue: no filtering of the components through the swab or fabric.

Some lateral flow tests have a second 'pad' built into the cassette, in essence they have their own filter: many diagnostic tests have this as they are designed to handle raw body fluids directly. Forensics is different and raw body fluids are rare and we manufacture our tests without this second pad and therefore require extracts from swabs or fabric.