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Human Blood Infidelity Test

The immunoassay enables the user to identify traces of human blood in forensic sample material. In a specific antigen/antibody reaction, the presence of human hemoglobin can be determined within minutes. The test result is interpreted visually by the appearance of a red test result line in hemoglobin positive samples. The assay is rapid and easy to perform and requires no difficult manipulation of the sample material. If necessary, it can be performed directly at the crime scene.

Conventional methods for the detection of blood are often based on the detection of hemoglobin but do not allow the answer if the blood is of human origin. To answer this question additional testing is required. Frequently immunological tests or DNA-based hybridization assays are used, which are work-intense and time-consuming.

The sample is transferred into the extraction buffer in the collection tube and extracted/dissolved by shaking. After that three drops of the liquid are added directly to the test.

Results are interpreted visually 5 minutes after the addition of the sample. The lower detection limit is at least 40-ng/ml hemoglobin. This corresponds to human blood that has been diluted to 10-6 - 10-7 (average hemoglobin content of blood: 120-180 mg/ml). Due to the specificity of the antibodies, positive test results allow the direct conclusion that the sample most probably contains blood of human origin. A cross reactivity is observed for blood of primates.

The test contains two monoclonal anti-hemoglobin antibodies as active compounds. One of the antibodies has been immobilized as line in the test result region (T) of the membrane. The other antibody is gold-labeled. It is placed downstream of the membrane on a glass fiber pad and becomes mobilized by the addition of the liquid. If hemoglobin is present in the sample it will bind to the gold-labeled antibody forming a complex. Through the capillary effect of the membrane, the liquid moves across the membrane towards the test result region. Here the immobilized antibody that recognizes another epitope of hemoglobin will bind the hemoglobin complex with the gold-labeled antibody (formation of a sandwich complex). This can be seen by the appearance of the red test result line. The control line is composed of a different antibody pair (rabbit antibodies/goat anti-rabbit antibodies) one of which is immobilized at the control region. The other one is gold-labeled and mobile. As these antibodies interact directly with each other, the control line is formed independent of the presence of hemoglobin. The appearance of the control line serves as an internal control for the correct performance of the test.

A high dose hook effect is observed, if too much free hemoglobin that is not bound to the gold-labeled antibody reaches the test result region. In this case the antibody immobilized at the test result region becomes saturated with free hemoglobin. This prevents the binding of the hemoglobin complex with the gold-labeled antibody, thus interfering with the formation of the test result line. The test result appears negative in spite of the presence of hemoglobin in the sample. The high dose hook effect can be avoided using the color of the sample as a guide. The visual detectable color caused by hemoglobin vanishes between a 10-3 and 10-4 dilution. At this concentration range there is no danger of a high dose hook effect. In contrast samples that are clearly colored due to hemoglobin are likely to cause false negative results because of the high dose hook effect.

Intended use: Detection of human hemoglobin in forensic case material Indication: suspected presence of human blood/confirmation of the human origin of bloodstains Material tested: extracted blood stains, blood (use appropriate dilution to avoid high dose hook effect) Principle: chromatographic sandwich immunoassay Sensitivity: 40 ng/ml hemoglobin (lower detection limit) 500 µg/ml hemoglobin (upper detection limit) Test procedure: test is started by the addition of 3 drops of liquid into the sample well Time required: 5 minutes at room temperature, negative test results should be confirmed after 10 minutes Interpretation: visual; positive test results are indicated by the formation of a red test result line Storage: refrigerated or at room temperature (+4-30 °C) Specificity: cross-reactivity with blood of primates, no cross reactivity with bovine, dog, rabbit, cat, pig, wild boar, horse, chicken, sheep, mule or red deer hemoglobin

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