Antibody production

Dom Spinella dspinella at
Thu May 29 16:52:41 EST 1997

D.L. Roberts writes:

> Hi,
> We have recently been trying to produce a polyclonal antibody against 
> caspase-10 (large subunit) an enzyme involved in apoptosis and we have 
> encountered a problem. The rabbit was injected (sub-cut) with 1mg of 
> protein and the titre of the antibody was followed for 70 days and the 
> titre peaked but did not decrease at all. The rabbit was boosted at day 
> 70 with 1mg of the antigen in the same manner. It is now 30 days later 
> and the titre is dropping but the antibody still is very poor when used 
> for western blotting. Could anyone suggest anyways of improving the 
> titre / quality of the antibody? One suggestion to date is an IV 
> injection of the antigen would this help?
> Thanks for any advice 

You don't mention the use of any adjuvant. Its been quite afew years,
but in my experience, the best immunization regimen for producing
high-titered antiserum in rabbits is to inject sub-cut (or IM) with
antigen emulsified in Complete Freund's Adjuvant (CFA). This can be
followed up with a couple of boosters at 2-3 week intervals using the
same antigen in Incomplete Freund's. You can sometimes further increase
the titer with an IV injection (without adjuvant) 7-10 days after the
last boost. CFA is a bit hard on the bunnies (sometimes causing sterile
ulcers) and many animal facilities now frown on its use. At least one
company (RIBI) claims to have a good substitute, but I have never used

By the way, I don't know how you are purifying your enzyme, but if
acrylamide gel electrophoresis is useful, it turns out that polymerized
acrylamide is also a pretty good adjuvant. You don't need to elute from
the gel -- simply cut out the appropriate band, crush it up and emulsify
an CFA or IFA, and inject.  Works great!

The final issue to consder is how different the rabbit version of the
antigen you are using is from waht you are working with.  Animals are
generally tolerant to self molecules, and the greater the difference
between the injected enzyme and the autologous version in rabbits, the
stronger your immune response will be.  If bunnies don't respond well,
you can try guinea pigs or rats (or even goats or chickens) if they are
accesible to you.

Hope this helps.
D.G. Spinella

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