polyclonal antibody production protocol - looking for protocols (Jul/24/2007 )
Hello, i am looking for the protocol to produce rabbit polyclonal antibodies against human antigens. I was wondering what is are the suitable techniques? if anyone can provide me sources of protocols it would be very much appreciated.
There are a few parts to producing a polyclonal antibody. These include:
-Determining your antigen sequence
-Cloning antigen sequence into appropriate vector
-Expressing and purifying antigen (i.e via His tag)
-Preparation of injection and injection schedule
-Analysis of antibody in serum
When I made antibodies I contacted the Animal House of my institution to request any guidelines they had concerning injection schedules and preparations, in particular requesting information on current rabbit protocols they were using. This way you can make sure you are not harming the animal and are acting according to your institution recommendations as every protocol may not be suitable for you.
I am assuming you have a purified antigen already.
This is a protocol I have written & used:
****Remember to ask for a pre-injection bleed so that you have rabbit serum for a negative control. You will be injecting the animals (normally two animals per antigen) at 1-6 weekly intervals for a period of 3-4 injections. You will request a test bleed between these injections to test the antibody titre.
Preparation of Antigen
Aliquot the appropriate volume for 500ug** antigen and centrifuge to pellet the precipitate.
(note here that I say precipitate because my purified antigen was insoluble. An antigen can be soluble too)
** 500ug was used for first inj, then 200ug for follow up inj.
Remove the supernatant and resuspend the pellet in 300ul sterile TBS.
Sonicate the antigen/TBS for 1min using the water bath sonicator in the Proteomics Labs. This will resuspend any obvious precipitated antigen clumps.
Have the remaining 700ul sterile TBS per rabbit ready for preparation of the adjuvant/antigen emulsion.
Preparation of Antigen:Adjuvant Injection: Maximum 2ml per rabbit.
Adjuvants are irritants and should be prepared with care.
CFA is a water-in-oil emulsion containing killed mycobacteria while IFA does not include mycobacteria. CFA is given as an initial dose to prime the animal, and IFA as subsequent injections. CFA must never be used more than once in an animal.
1. Obtain 2 x 5ml sterile Luer Lock Syringes and 1 x BD Connecta Luer Lok (REF394600).
2. Sigma’s Complete Freunds Adjuvant (CFA) (F5881) and Incomplete Freunds Adjuvant (F5506) are required for the first and subsequent injections, respectively. The CFA must be mixed thoroughly prior to use to ensure even distribution of mycobacteria.
3. Draw 1ml FA per rabbit into the sterile Luer-Lok syringe.
4. Draw a portion of the aqueous component (the 300ul resuspended antigen per rabbit) into the syringe containing the total volume of FA. Remove excess air from the syringe.
5. Connect the syringe to the first port of the sterile three way tap, where an empty syringe occupies the second port.
6. Force the adjuvant / antigen solution through repeatedly between the two syringes.
7. A second portion of the aqueous component is then added as above until all the aqueous component (700ul per rabbit) has been incorporated into the emulsion.
8. Continue mixing until the emulsion becomes milky and then pasty. Note: Emulsification is enhanced by use of cold (4C) adjuvant. Insufficient mixing is a major cause of immunisation failure.
9. Keep the syringes connected to the three way tap and take down to the Animal House
Preparation of Serum
1. Place blood at room temperature for 1 hour.
2. Loosen clot from side of tube using a pasteur pipette.
3. Incubate overnight at 4ºC.
4. Remove serum and centrifuge at 4000xg for 10min at 4ºC.
5. Store serum at 4ºC for immediate use OR aliquot and store at -20ºC
to shorten the procedure, which is time consuming anyway. I would suggest to let synthesize and modify (carrier-coupling) your peptides of interest by a company. then start with immunization...