42. Immune reactions with labeled antibodies.

Immune reactions with labeled antibodies.

• Definition and Purpose: Labeled antibodies are antibodies that have been conjugated, or linked, with a detectable marker or label. Their purpose is to detect and quantify specific antigens or antibodies in biological samples by making the antibody-antigen interaction visible or measurable.
• Types of Labels Used: Common labels include fluorescent dyes, enzymes, radioactive isotopes, and biotin.
• Key Techniques Employing Labeled Antibodies: These techniques utilise the labeled antibodies to visualise or measure the outcome of an antigen-antibody reaction:

• Immunofluorescence (IF): Uses fluorescent labels (e.g., FITC, PE).
• Direct Immunofluorescence: A primary antibody, directly labeled with a fluorescent dye, binds to the target antigen in the sample. The presence of fluorescence indicates the antigen is present.
• Indirect Immunofluorescence: A primary antibody (not labeled) binds to the target antigen, and then a secondary antibody (labeled with a fluorescent dye) binds to the primary antibody, amplifying the signal.
• Applications: Diagnosis of viral infections such as herpes simplex virus (HSV) using tissue biopsy samples. Detection of Legionella antigens or antibodies.

• Enzyme Immunoassay (EIA) / ELISA (Enzyme-Linked Immunosorbent Assay): Uses enzyme labels (e.g., Horseradish peroxidase, Alkaline phosphatase). The enzyme converts a substrate into a detectable signal, often a colour change.
• Different formats exist, including Indirect ELISA (detecting antibody), Sandwich ELISA (detecting antigen), Competitive ELISA (detecting antigen), and Capture ELISA (e.g., for detecting IgM antibodies).
• Applications: Quantification of antigens or antibodies. Diagnosis of diseases such as HIV and Hepatitis, cancer biomarkers, Legionella antigen detection in urine, and detection of IgM antibodies in viral infections. Also used in Western blotting and solid phase tests.

• Radioimmunoassay (RIA): Uses radioactive isotopes as labels. The interaction between the labeled antibody/antigen and its target is measured by the emitted radioactivity.
• Applications: Quantification of antigens (e.g., hormones, drugs) or antibodies (e.g., autoantibodies) in biological samples. Examples include Radioallergosorbent Test (RAST) and Radioimmunosorbent Test (RIST) for detecting specific IgE antibodies.

• ELISPOT Assay: Utilises enzyme-coupled antibodies fixed on a surface to capture secreted products like antibodies or cytokines from cells incubated on the surface. Spots of colour indicate the location and frequency of secreting cells.
• Flow Cytometry: Analyzes cells using fluorescently labeled antibodies that bind to cell surface or intracellular antigens (e.g., CD markers). Allows for identification and quantification of different cell populations.