By R. Edwards (Editor)
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Extra info for Immunodiagnostics: A Practical Approach (Practical Approach Series)
Edwards et al. • lactoperoxidase, usually coupled to a solid-phase • chloramine T • iodogen Using lactoperoxidase is considered to give the most mild oxidation and thereby greatly reduces the possibility of damaging the tracer molecule. Nonetheless, the simpler techniques using chloramine T or iodogen have been used successfully to prepare many and perhaps the most commonly used radiolabels. The protocols given have been optimized to yield a product with approximately 1 atom of radioiodine per molecule of tracer.
The F(ab)2 is divalent as it retains both binding sites in one fragment. Conventionally the enzyme pepsin is used to digest immunoglobulin molecules to give F(ab)2 and papain for Fab and Fc fragments. It is important to note that different classes of immunoglobulins differ in their sensitivity or resistance to enzyme digestion. This is also true for classes or subclasses from different species of animals. For example, sheep immunoglobulins are more resistant to pepsin than those from rabbit. Subclasses of mouse immunoglobulins differ widely in sensitivity to pepsin, in particular with respect to myeloma antibodies, viz.
Preparation of immunosorbent 1. Add CNBr activated Sepharose 4B in 1 mM HCI (approximately 1 g in 10 ml) to make a slurry and wash on sintered glass filter with a further 190 ml over 15 minutes. 23 R. Edwards el al. Protocol 14. Continued 2. 0). NB. No azide in buffer. 3. Add antigen solution to swollen gel and mix gently (by rotation) for 2h. 4. Separate gel on glass filter. 5. Add 1 M ethanolamine pH 8 (Merck) in coupling buffer and mix gently for a further 2 h. 6. Repeat separation step to remove ethanolamine solution.
Immunodiagnostics: A Practical Approach (Practical Approach Series) by R. Edwards (Editor)