Antibody Modification and Conjugation

The heavy chains of each immunoglobulin molecule are identical. Depending on the class of immunoglobulin, the molecular weight of these subunits ranges from about 50,000 to around 

Similarly, the two light chains of an antibody are identical and have a molecular weight of about 25,000. For IgG molecules, the intact molecular weight representing all four subunits is in the range of 150,000-160,000. 

There are two antigen binding sites on each of the basic Ig-type monomeric structures, formed by the heavy-light chain proximity in the N-terminal, hypervariable region at the tips of the y structure. The unique tertiary structure created by these subunit pairings produces 

The major enzymes used in ELISA technology include horseradish peroxidase (HRP), alkaline phosphatase (AP), (3-galactosidase (P-gal), and glucose oxidase (GO). See Chapter 26 for a detailed description of enzyme properties and activities. HRP is by far the most popular enzyme used in antibody-enzyme conjugates. One survey of enzyme use stated that HRP is incorporated in about 80 percent of all antibody conjugates, most of them utilized in diagnostic assay systems. 

AP is the second most popular choice for antibody-enzyme conjugation, being used in almost 20 percent of all commercial enzyme-linked assays. Although P-gal and GO are used frequendy in research and cited numerous times in the literature, their utilization for commercial ELISA applications represents less than 1 percent of the total assays available. 

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