Unit 9: Immunochemical Techniques

1. Introduction

This discussion is kept to a minimum because the appendices provide a wealth of details about the use of antibodies in biotechnology. Antibodies are used to detect antigens in the following scenarios: ( i) in solutions, ( ii) in gels, ( iii) on blotting membranes, ( iv) during or after attachment to affinity chromatography matrices, and (v) in situ and in vivo within cells, tissues cells, systems, and organs. An "immunoassay" involves using an antibody to detect an antigen. In the "simplest" situation, the antigen is detected by preferential retention of antibody-antigen complexes to solvent filters or blotting membranes; antibody-free antigen is not retained. If the antibody is recognized, the protein binds the antigen and the complex adheres to the filter or "blot" matrix.

2. Antibody and Antigen Labeling; Detection Strategies

One "labels" either the antigen or the antibody. "Labeling" involves several types of processes; a range of examples is given to illustrate some typical applications.

In one approach, the antigen is labeled directly with a radioisotope, fluorophore, or ligand that can be "grabbed" by a subsequent reagent in the assay protocol. The isotope is detected by exposure to a film or image storage plate. Fluorophores can be imaged by exciting them with light at the appropriate absorption wavelength and measuring the fluorescent pattern at the emission wavelength. Such images have been obtained with aqueous samples in Eppendorf tubes, using antigens that have been separated by electrophoresis or chromatography, and...