Techniques for Species Identification

Once a suspected stain is confirmed to be blood, it can be further examined to identify if the blood is of human origin. If a bloodstain is determined to be of non-human origin, it is usually not necessary to conduct further analysis, as it is unlikely to be relevant to the investigation. Eliminating non-human samples can save time and resources, leading to more efficient and cost-effective investigations. Serological techniques used for species identification can be classified into primary and secondary binding assays, discussed below:

Figure: Techniques for Species Identification

1. PRIMARY BINDING ASSAYS

Primary binding assays, also known as direct assays, directly measure the binding of the target analyte (an antigen or antibody) and a labelled probe. The label is typically an enzyme or fluorescent molecule that can be detected to measure the presence or concentration of the target analyte.

For example: Enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), and immunochromatographic assays.

📌 Read more: Primary Binding Assays

2. SECONDARY BINDING ASSAYS

Secondary binding assays, also known as indirect assays, measure the result (such as precipitation or agglutination) of the antigen-antibody reaction rather than directly measuring the binding. This can involve detecting the formation of a larger complex or a change in the physical properties of the solution after the primary interaction.

Secondary binding assays can classified into two categories:

A) Precipitation-based Assays

Precipitation-based assays are the most commonly used secondary binding assays that measure the formation of insoluble visible precipitation caused by the binding of soluble antigens and antibodies. They can be further categorized into:

1. Immunodiffusion Assays

   This technique is based on the diffusion of antigens and antibodies through a gel or agar medium. The antigens and antibodies form visible precipitation lines when they encounter each other, allowing for the identification and quantification of specific proteins.

   For example: Radial Immunodiffusion, Ring Assay, and Ouchterlony.

   📌 Read more: Immunodiffusion Assays

2. Immunoelectrophoretic Assays

   This technique combines immunodiffusion with electrophoresis. encompasses two principles: firstly, an electrophoresis for separation of different antigens in agar medium, followed by an immunodiffusion against the antiserum resulting in precipitin formation. This provides increased sensitivity, improved specificity, and the ability to detect multiple antigens in a single analysis.

   For example: Immunoelectrophoresis (IEP), Crossed Immunoelectrophoresis (CIE), Crossed-over Immunoelectrophoresis (COE), and Rocket Immunoelectrophoresis (RIE).

   📌 Read more: Immunoelectrophoretic Assays

B) Agglutination-based Assays

Agglutination-based reactions involve the clumping or aggregation of particulate antigens (very very small antigens) facilitated by antibodies. This occurs when antibodies help in cross-linking multiple antigens together, resulting in the formation of larger, visible aggregates in the solution.

1. Direct Agglutination: Direct agglutination is a type of agglutination assay where antibodies directly interact with particulate antigens, leading to visible clumping or aggregation. The most common application of direct agglutination is ABO blood typing, where RBCs act as the particulate antigens. The agglutination of these RBCs in the presence of specific antibodies can help in determining the blood type of an individual.

2. Agglutination Inhibition: Agglutination inhibition assays are a type of immunoassay designed to detect the presence of specific antigens in a sample by inhibiting the agglutination or clumping of particles. Agglutination inhibition is useful for detecting soluble antigens, such as hormones or certain pathogens, which may not cause visible agglutination in a direct agglutination assay.

3. Passive Agglutination Assay: Agglutination inhibition assays are a type of immunoassay designed to detect the presence of specific antibodies in a sample. Particles are sensitized with target antigens, and agglutination occurs when antibodies in the sample bind to these antigens. Passive agglutination assays are commonly used to detect the presence of antibodies for Rh factor (Rh antigen, also known as antigen D) for identifying potential Rh incompatibility between a pregnant woman and her unborn child.

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