Mouse immunization protocol - how to improve the affinity of immune response (Nov/20/2007 )
Hi,
another question is how to immunize mouse properly, in case you have conservative protein as an antigen.
We perform classic immunization protocol:
First immunization, s.c. 50 microgramm of conjugated haptene, or 30 micro of protein in 150 microl total with Freund`s complete adjuvance.
After 10 days, we repeat this with incomplete adjuvance.
After next 10 days we repeat it again third immunization.
And after next 10 days fourth immunization we perform next and last immunization i.p. twice of antigen concentration.
We perform fusion 4 days after final boost.
The question is in which way we can improve the mouse immune responce.
If there are any ways to improve it, it would be great to know it. And if there are people working in the field of mouse immunology, they for sure will know better, the knew and relyable ways to perform better.
I mean, I`ve read about different antigen concentrations, and immunizing with lower at the end activate only highly specific B-cells. I don`t know much about time coarse and variation with it.
The next thing, that can improve immune responce might be, conjugate protein ( now we use KLH, before we used BSA or OVA)
I thank everybody in advance for any responces, it is simply not the case that we can verify all the things by ourself.
Andrej
Hi,
Just before the last immunization you can extract blood, from the tail vein, to perform an ELISA to obtain the antibody titer. This can permit you decide to use one or other mouse in the cell fusion (you may have different titles of antibody), or even extend immunization schedule if you see that the answer is low, increasing the dose of antigen target.
If the title of antibodies in blood is bad, it is very difficult to obtain hybridomas and monoclonal antibody, and it is almost better not to proceed with the cell fusion.
tonix
This is the protocol I used, and it worked really nice.
We precipitate proteins with alum for immunisation. Briefly, one volume of 9 % potassium alum (Sigma, aluminium potassium sulphate) 1s added to the protein solution at 0.5 mg/ml. Phenol red indicator is then added, and the solution neutralised by adding 1 M NaOH until the indicator turns pink (takes quite a bit of NaOH). Proteins are allowed to precipitate at room temperature for 30 min with rotation. After this time, wash the precipitated protein 3 times with PBS, until it becomes white. Mice are injected subcutaneously on day 0 with 100 µg. Mice are then challenged intravenously with 1 µg of protein (alum precipitated) on days 28, 29 and 30. Day 31 mice were sacrificed and serum and spleens were collected.
L
hi,
during second and third immunisation reduce the quantity of protein to half.
this will increase the specificic clonal expansion.
u can find hybridoma which release highly specific antibodies after fusion.
gud luk
sravan.
during second and third immunisation reduce the quantity of protein to half.
this will increase the specificic clonal expansion.
u can find hybridoma which release highly specific antibodies after fusion.
gud luk
sravan.
Thanks for the advice. Competely agree with you, I did it in summer, and actually have had indeed better results. I am also interested in intervals between immunizations to improve specificity of antibody. Any ideas?
I have now two different protocols. One is that you immunize after 10 days then third after 10 days, then forth after ten days.
Another is that you can immunize week after first immunization, then one more week, and the forth immunization min after 2-3 weeks. Something better?
We precipitate proteins with alum for immunisation. Briefly, one volume of 9 % potassium alum (Sigma, aluminium potassium sulphate) 1s added to the protein solution at 0.5 mg/ml. Phenol red indicator is then added, and the solution neutralised by adding 1 M NaOH until the indicator turns pink (takes quite a bit of NaOH). Proteins are allowed to precipitate at room temperature for 30 min with rotation. After this time, wash the precipitated protein 3 times with PBS, until it becomes white. Mice are injected subcutaneously on day 0 with 100 µg. Mice are then challenged intravenously with 1 µg of protein (alum precipitated) on days 28, 29 and 30. Day 31 mice were sacrificed and serum and spleens were collected.
L
Wow!!! really nice protocol, I haven`t heard about . Great info!!!
Just before the last immunization you can extract blood, from the tail vein, to perform an ELISA to obtain the antibody titer. This can permit you decide to use one or other mouse in the cell fusion (you may have different titles of antibody), or even extend immunization schedule if you see that the answer is low, increasing the dose of antigen target.
If the title of antibodies in blood is bad, it is very difficult to obtain hybridomas and monoclonal antibody, and it is almost better not to proceed with the cell fusion.
tonix
thanks for advices too. We do analysis of tail vein blood from time to time, generally we don`t have different titres, only once from 15 mices I could not see immune reaction. Agree also with you, that if the titre of antibodies is low, its better to start another immunization, and not waste the time on fusion and cloning