Immunochemical Protocols (Methods in Molecular Biology) by John Pound

By John Pound

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Extra resources for Immunochemical Protocols (Methods in Molecular Biology)

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Wang Laboratory of Lymphocyte Molecular Biology, Imperial Cancer Research Fund, London, UK Wendy W. , Eugene, OR Page 1 1 Production of Polyclonal Antisera Jonathan A. Green and Margaret M. Manson 1 Introduction All immunochemical procedures require a suitable antiserum or monoclonal antibody raised against the antigen of interest. Polyclonal antibodies are raised by injecting an immunogen into an animal and, after an appropriate time, collecting the blood fraction containing the antibodies of interest.

1 Immunizations 1. FCA and FIA: FCA is a suspension of killed pertussis in oil. This should be shaken well before use. FIA is just oil. Freund's adjuvants should be mixed with the aqueous immunogen solution/suspension at a ratio between 1:2 and 2:1 and mixed until set. This is most easily achieved by passage back and forth between two glass syringes connected by a three-way luer fitting. After a while (1-5 min) the mixture should become noticeably "stiffer," and may then be used. 2. Potassium aluminum sulfate (Alum) AIK(SO4)2.

Up to four rabbits about 4-6 mo old. Various strains can be used, including half sandy lops or New Zealand whites (see Note 1 ). 3 Method 1. Take up 1 mL of complete adjuvant in one of the syringes and 1 mL of antigen solution containing approx 100 mg of the antigen in another. Attach both to the plastic connector (Fig. 1), making sure that the tap on the connector is open in such a way that only the two ports connecting the two syringes are open. Repeatedly push the mixture from syringe to syringe until it becomes thick and creamy (at least 5-10 min).

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