Polyclonal Antibody Production is a quick and easy way to prepare multi-resistance methods for use in Western, ELISA, and immune/b.

Very useful for rapid and simple generation of antibodies for western blots, ELISA assays, and immunoprecipitation.

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Initial Preparation

Your antigen should at least have a molecular weight of 20 kDa (kilodaltons) for development of a strong immune response in animals. If it is a small protein, it can be linked to a larger protein by chemical methods or cloned and expressed as a fusion protein with a larger protein. If it is a peptide or small molecule, a carrier-hapten conjugate must be made. Purify your antigen well to obtain antibodies with good specificity.

Obtain at least two 6 week old New Zealand White rabbits and have them delivered to the Animal Resources Center. Don't put all your eggs in one basket by using only one rabbit. Allow them to get accustomed to their new environment for two weeks before the first pre-bleed.

Pre-bleed

Calmly and gently place rabbit in a restrainer. If they are relaxed they bleed much easier.

For injection of two rabbits, your antigen should be in 1 mL of a buffer such as phosphate buffered saline (PBS) or Tris that contains no chemicals harmful to the rabbit. 100 µg/rabbit of antigen is best, but you can use less and get good results.

The quality of the antibodies in serum (humoral immune response) of the bleeds is monitored by indirect ELISA. Titer is defined as the dilution that gives you 1/2 the maximal absorbance of this assay.

Purification of