General rabbit polyclonal antibody production protocol:

1) Immunization animal: Select one healthy New Zealand rabbit. 

2) Adjuvants: Start with Complete Freund’s Adjuvant (CFA), followed by Incomplete Freund’s Adjuvant (IFA). 

3) Antigen: Protein or KLH-conjugated peptide. Immunize with 100–200μg of antigen each time. 

4) Immunization: Dilute the antigen with saline, then mix 1:1 with the corresponding adjuvant. The antigen and adjuvant are thoroughly mixed to form a stable emulsion, draw the antigen mixture with a syringe, and inject the antigen at two subcutaneous points on the rabbit’s shoulders and two points into the muscles of both hind legs. About 1/4 volume of the antigen is administered per area. This way, the antigen persists for a longer time to enhance the immune response. 

5) Blood collection: Blood is drawn from the rabbit’s ear artery using a 19-gauge needle, left at room temperature overnight for serum separation. Day 1 Immunization 1: Collect 5ml of whole blood (to obtain 2–3ml pre-immunization serum) left at room temperature overnight, centrifuge the next day and aliquot. 

6) Immunize the rabbit with 200μg of antigen mixed with Complete Freund’s Adjuvant (CFA);  

7) Immunization 2(Day 15): Immunize the rabbit with 200μg of antigen mixed with Complete Freund’s Adjuvant (CFA);  

8) Immunization 3(Day 29): Immunize the rabbit with 100μg of antigen mixed with Incomplete Freund’s Adjuvant (IFA); Day 43 Collection 1: Collect 5ml of whole blood for ELISA or WB testing;  

9) Immunization 4(Day 44): Immunize the rabbit with 100μg of antigen mixed with Incomplete Freund’s Adjuvant (IFA); if Collection 1 ELISA is positive, start collecting serum, draw 30ml of blood to collect about 15ml of serum; 

10) Day 58 Collection 2: If Collection 1 ELISA is negative, still take a small sample (as in Collection 1), send to the laboratory and wait for the result;

11) Immunization 5, Collection 3(Days 59, 73… ): Continue as such, drawing blood every two weeks, if ELISA is positive, start collecting serum, collect serum 5 times (totaling 70–80ml serum) after the rabbit becomes positive. 

Note: During the entire project, the rabbit needs to maintain 10 weeks of immunization and bleeding. The results of the tests will determine the continuation, modification, or termination of this project.

12) Purification: Peptide affinity purification of antibodies or Protein A/G purification of antibodies. Testing: Antibody ELISA, WB, IHC, and IF application validation.

The immunization protocol might be changed accroding to different applications and requirements. Alpha Lifetech Inc.’s custom antibody development services including monoclonal and polyclonal antibody generation, production and modification services. We provide a wide range of polyclonal antibody production in different species including New Zealand white rabbits, goats, sheep, guinea pigs, chickens, rats, mice and donkeys.