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.

  

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.

  

  

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.

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.

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.

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.

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.