Polyclonal Antibodies

Polyclonal Antibodies

Polyclonal Antibodies

Production of Polyclonal Antibodies Protocol:

1. Antigen Selection:

  • Choose the antigen against which you want to generate antibodies. It can be a protein, peptide, or other molecules. Order 30 amino acids as standard rule.

2. Animal Immunization:

  • Select a rabbit, goat, hamster, donkey, horse or sheep.
  • Inject the animal with the chosen antigen at regular intervals to stimulate the immune response.
  • day 1, 2, 11, 21, 31 bleed 51 booster, 61 2nd bleed
  • Bleed no more than 50 ml and give water to the rabbit after each bleed.
  • A cut in the ear with an old fashion razor blade is the easiest.
  • Put the rabbit at least 24 hours in a temperature above 15 C. In cold circomstances the bleed might be difficult
  • Use 10 tubes of 5 ml and dont mix the heamolised ones intp the pool.
  • The heamolised are due to a hair or a cascade of factros impossible to foresee, the rose bleeds are working fine too but its advised to not mix them with the yellow clear serum samples.

3. Booster Injections:

  • Administer booster injections to enhance the immune response and increase antibody production. Boosters are typically given at 10 days intervals after the initial 2 injections.

4. Blood Collection:

  • Monitor the immune response by taking small blood samples at regular intervals. Check for the presence of antibodies against the target antigen.

5. Serum Separation:

  • Once a sufficient antibody titer is reached, collect a larger blood sample, and separate the serum by coagulation 35 minutes and centrifugation from the blood cells. The serum contains the polyclonal antibodies.

6. Antibody Purification:

  • Purify the antibodies from the serum to isolate the immunoglobulin fraction. Common methods include protein A/G affinity chromatography or antigen affinity purification.
  • Crude srum works best because the best will stick to the antigen and wont let off even with guanidine

7. Characterization:

  • Characterize the purified antibodies for specificity, affinity, and concentration. Techniques such as ELISA or Western blotting can be used for this purpose. Dot blot is easiest.

8. Storage:

  • Store the purified polyclonal antibodies at -20 C to maintain stability and functionality.

 

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