Abstract
The scope of this book does not allow a complete description of the many techniques available for improving and facilitating immunoassays in general. There is a large amount of literature covering techniques, and these can be consulted for specific problems. Examination of many of the catalogs produced by commercial companies is useful, since they often include good technical sections describing methods using their products. This chapter contains the practical basics of conjugation (a large field in itself) and details of other immediately useful techniques that might be first desired in starting ELIS A. The book recommended (1) should be considered definitive in the laboratory since it is extremely “digestable” and covers a large field of methods, all of which are relevant to ELISA.
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Notes
- 1.
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Use a gel matrix with an exclusion limit of 20,000–50,000 for globular proteins. Use medium-sized beads (approx 100 μm in diameter). Prepare a column with 5 mL of bead volume according to the manufacturer’s instructions. To make the column easier to load and run, first add 20 μL of glycerol and 20 μL of 1% xylene cylanol. The column should be prerun with a minimum of 10 column volumes of 0.15M NaCl. Allow the column to run until the buffer level drops just below the top of the bed resm. Stop the flow of the column. Carefully load the column with the glutaraldehyde-treated HRP. Release the flow, and allow the HRP to run into the column. Just as the level of the HRP solution drops below the top of the column, carefully add 0.15M NaCl. Run the column with 0.15M NaCl. Pool the fractions that look brown. These contain the active enzyme
References
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Immunogens, Ag/Ab Purification, Antibodies, Avidin-Biotin, Protein Modification: PIERCE Immunotechnology Catalog and Handbook, vol. 1. Pierce and Warriner, UK Published yearly.
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Crowther, J.R. (1995). Immunochemical Techniques. In: Crowther, J.R. (eds) ELISA. Methods in Molecular Biology™, vol 42. Humana Press. https://doi.org/10.1385/0-89603-279-5:207
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DOI: https://doi.org/10.1385/0-89603-279-5:207
Publisher Name: Humana Press
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Online ISBN: 978-1-59259-529-7
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