Chapter 13: Species Identification

13.1: General Considerations

Types of Antibodies

  • An antihuman antibody that is used in the identification of human samples can be made by introducing human serum into a host animal, which then produces specific antibodies against the human serum proteins.
  • Antibodies produced from different species of host animals may produce variations in the characteristics of reactions.
  • Albumin: A protein that plays important roles in the maintenance of the vascular circulating fluid and the transportation of various substances such as nutrients, hormones, and metabolic products.
  • Hemoglobin: An oxygen-transport protein that is found in erythrocytes.
  • Purified Hemoglobin: Can be used to generate monoclonal and polyclonal antihuman Hb antibodies.

Titration of Antibodies

  • An extreme excess of antigen or antibody concentrations can inhibit secondary reactions.
  • The prozone and postpone phenomena must be considered, and the concentrations of antigen and antibody must be carefully determined for forensic serology assays.
  • Quality-control procedures can be used to estimate the amount of a specific antibody that is present, often via titration.
  • To titrate an antiserum, a series of dilutions are made and each dilution is then tested for activity using precipitation or agglutination methods.
  • Titer: The reciprocal of the highest dilution giving a positive reaction.

   Titration of antibodies. Serum is serially diluted and a constant amount of antigen is applied to each tube. The mixture is incubated, allowing agglutination to occur.

Antibody Specificity

  • The specificity of the antihuman antibody must be tested.
  • Most anti-human antibodies usually have cross-reactivity with higher primates. This is not a great concern because crimes involving nonhuman primates are very rare.
  • The anti-human antibody must not cross-react with other commonly encountered animals.

Optimal Conditions for Antigen–Antibody Binding

  • Stronger inhibition is usually observed for ions with large ionic radii and small radii of hydration.
  • A proper buffer system must be selected in serological assays to ensure reliable results.
  • The introduction of polymers can facilitate precipitation in a a secondary binding reactions because the presence of a polymer in a solution decreases the solubility of proteins. Linear hydrophilic polymers with high molecular weights are preferred.

13.2: Assays

Immunochromatographic Assays

  • These are rapid, specific, and sensitive and can be used in both laboratory and field tests for species identification.
  • Hexagon OBTI and ABAcard HemaTrace® can utilize the antibody–antigen–antibody sandwich method by using antibodies that recognize human Hb.   * ABAcard HemaTrace®: It utilizes a labeled monoclonal antihuman Hb antibody contained in a sample well, and a polyclonal antihuman Hb antibody immobilized at a test zone of a nitrocellulose membrane.
  • RSID™-Blood use antibodies that recognize human GPA.

   Immunochromatographic assays for the identification of Hb in human blood.

   Diagram of the structure of glycophorin (GPA) protein.

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Double Immunodiffusion Assays

  • Ring Assay: An antihuman antibody reagent is placed at the bottom of a test tube and a blood-stain extract is placed on top of the bottom layer.
  • Ouchterlony Assay   * In a positive reaction, a line of precipitate will form between each antigen well and antibody well. This assay can also determine the similarity of the antigens.   * In an assay in which two antigens are loaded in adjacent wells and an antibody in the third well, the following results can be observed:     * Identity: A phenomenon wherein two antigens are identical, the two lines will become fused.     * Nonidentity: A phenomenon wherein two antigens are totally unrelated, the lines will cross each other but not fuse.     * Partial Identity: A phenomenon wherein the two antigens are related but are not identical, the lines will merge with spur formation.

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Ring Assay Procedure
  • Sample preparation and extraction   * Extract a portion of a stain with saline at 4°C overnight.
  • Controls   * Include a positive control (known human serum sample) and a negative control (extraction blank).
  • Loading of antibody and samples   * Spin the antihuman antibody in a microfuge and transfer the supernatant into test tubes or capillary tubes (depending on the volume of the stain and the antiserum extracted).   * Place the sample carefully over the top of the antiserum solution, which is usually denser than the sample.
  • Immunodiffusion reaction   * Carry out the reaction at room temperature.   * In a positive reaction, white precipitate between the two layers can be observed after several minutes.     * This indicates that the sample is of human origin.     * No precipitate is formed if a bloodstain extract is from a nonhuman origin.

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Ouchterlony Assay Procedure
  • Sample preparation and extraction   * Cut out a small portion (approximately 5×5 mm) of a stain or a portion of a swab.   * Extract at room temperature in 100 μL of water for 30 min.     * The extract can be diluted if necessary.     * Alternatively, a very small piece of the stain or swab can be inserted directly into the well.
  • Controls   * Positive (known serum)   * Negative (extraction blank)   * Substrate controls (extraction of substrate from unstained area) if applicable
  • Agarose gel preparation   * Heat a suspension of agarose (4%) until liquefied.   * Cool the solution in a water bath at 55°C.   * Pour the agarose onto a piece of glass slide and let the gel solidify to a thickness of about 2–3 mm.   * Alternatively, a polyester support film such as GelBond (Cambrex, New Zealand) can be used as a gel support.   * The agarose should be poured onto the hydrophilic side of a piece of GelBond film (6×9 cm).   * Punch wells consisting of a central well surrounded by four wells using a template.
  • Loading antibodies and samples   * Apply antihuman antibody to the central well. Apply the positive control to one of the surrounding wells.   * Apply the sample(s) in question next to a positive control.   * Apply negative and substrate controls to the remaining wells; only one negative control is needed per gel.
  • Immunodiffusion reaction   * Incubate the plate overnight in a moisture chamber at 37°C.
  • Staining   * Soak the gel overnight in saline solution and then soak it in deionized water for 10 min. Repeat once.   * Dry the gel between paper towels with a weight on top for 30 min.   * Dry in an oven for 30 min.   * Stain the gel with Coomassie blue. Stained precipitate bands appear blue.

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Crossed-Over Electrophoresis

  • This method is a combination of immunodiffusion and electrophoresis.
  • A sharp precipitate band is visualized in a positive reaction.
  • False-negative results can occur due to the post zone phenomenon, in which excess antigen may inhibit precipitation.
  • False-negative results can also occur due to simple mistakes made during electrophoresis:   * Electrophoresis is carried out in the opposite direction, which results in samples running off the gel.   * Electrophoresis is carried out using an incorrect buffer system, affecting antigen–antibody binding.

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Crossed-Over Electrophoresis Procedure
  • Sample preparation and extraction   * Cut out a small portion (~5×5 mm) of a stain or a portion of a swab.   * Extract at room temperature in 100 μL of water for 30 min. The extract can be diluted if necessary.   * Alternatively, a very small piece of the stain or swab can be inserted directly into the well.
  • Controls   * Positive (known serum)   * Negative (extraction blank)   * Substrate controls (extraction of substrate from unstained area) if applicable
  • Agarose gel preparation   * Heat a suspension of agarose (4%) until liquefied.   * Cool the solution in a water bath at 55°C.   * Pour the agarose onto a piece of glass slide and let it solidify.   * Alternatively, a polyester support film such as GelBond can be used as a gel support.   * The agarose should be poured onto the hydrophilic side of a piece of GelBond (6×9 cm).   * Punch small wells (about 1–2 mm) in rows using a template.
  • Loading antibodies and samples   * Apply antihuman antibody in one row of wells.   * Apply samples in the other row of wells.   * Apply the positive, negative, and substrate controls
  • Electrophoresis   * Submerge the agarose gel in an electrophoresis tank in proper orientation.   * The wells containing antihuman antibody should be closest to the anode (positive electrode) and the wells containing samples should be closest to the cathode (negative electrode).   * During electrophoresis, the antibody in the antiserum should migrate toward the cathode while the antigen migrates toward the anode.   * Electrophoresis is carried out at 10  V/cm for 20 min.   * Staining Soak the gel overnight in a saline solution and then soak it in deionized water for 10 min. Repeat once.   * Dry the gel between paper towels with a weight on top for 30 min. Dry in an oven for 30 min.   * Stain the gel with Coomassie blue. Stained precipitate bands appear blue.

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