Hybridoma stability - Hybridoma (May/06/2004 )

hi,
i used to follow the following time coarse and quantity of antigen for immunization. these are approximate values.

age of mice - 2-4 months
primary imm - 10-50ug (depends on availability) with FCA
2-6 weeks
1st boost - 1/2 of the primary dose with FICA
2-3weeks
2nd boost - 1/2 of the first boost with FICA
2-4 weeks
IV - half or equal amouts amounts of primary immunization
IP - 5 times more than primary immunization quantity
3days
sacrifice the mouse n proceed with hybrdoma procedure

Keep in mind that different proteins has got differnt immunogenicity.

hope this will help you

gud luk
sravan k. payeli

i m sorry, i forgot to mention the Ab titres of mice in my previous post.
i took approximately 0.8-1 OD and control was around 0.05

hi all ! i agree with payeli. amount of antigen used to immunise is very important not too low not too high. usually antigen doses( total of all the booster as well as primary dose) should not exceed 500ug. excessive dose in a single immunization will result in a state of tolerance.
and low doses will not activate the immune system.
a good immunization shedule includes giving right amounts of antigen spaced at right intervals.

hi leelaram ! you said you are getting good antibody titres . i dont see any problem with your antigen concen. or immunization shedule. i think you are loosing your clones during screening.
tell me what secondary antibodies you are using during screening?

hi all ! i agree with payeli. amount of antigen used to immunise is very important not too low not too high. usually antigen doses( total of all the booster as well as primary dose) should not exceed 500ug. excessive dose in a single immunization will result in a state of tolerance.
and low doses will not activate the immune system.
a good immunization shedule includes giving right amounts of antigen spaced at right intervals.

Hi shiva keshava,
thanks for agreeing with me, since MABMAN and LEELARAM has same problem, i would like to propose one of my impression about their situation.
when they saw high titres of antibody, may be those are igMs(in general this class showup early n in high titres n lost early).
i think, the time gap for first boost they give is not sufficient. that is the reason, they r stimulating the activated B cells, which produce high titres of IgM antibody for immediate clearance.
But we need memory cells to fuse with myeloma cells so that, they produce anitbodies continuously.
so they should concentrate in improving differentiation of memory cells.

we should aware of the point, each animal will have different response to antigen.

kesava, if u think the explanation is quite logical then they can give a try. i appreciate ur comments.

thanks
sravan k. payeli

hi all ! i agree with payeli. amount of antigen used to immunise is very important not too low not too high. usually antigen doses( total of all the booster as well as primary dose) should not exceed 500ug. excessive dose in a single immunization will result in a state of tolerance.
and low doses will not activate the immune system.
a good immunization shedule includes giving right amounts of antigen spaced at right intervals.

hi payeli ! you are absolutely logical ! now i say that might be one possibility.but then they didnt mention what isotypes they are using while checking the titres.
it is very imortant to know the time schedule and dosage given by MABMAN and LEELARAM and at what intervals they are screening for titres and not to forget again the isotype they are checking while screening only then we can find a solution for the problem.
hope MABMAN and LEELARAM will post the above mentioned aspects.
in our lab we follow a short term immunization schedule of 21/2 months with 5 immunizations and it works well. we faced the same problem as MABMAN and LEELARAM but late learnt that we are loosing positive hybridomas while screening by not having screened for all the isotypes.

thankyou payeli for this discussion. it was great
have a nice time

hi payeli and shiva keshava,
thank you very much for your suggestions
i would like to mention all the immunization schedule to you people so that i will get enough
feedback from both of you
usually i immunize 6weeks old balb/c mice female with 50ug of antigen in CFA
after 2 weeks i give 50ug booster in IFA
after 3 weeks i give a 2nd booster of same dose and check the titre by
bleeding the mice after 10days of immunization
if needed i give another booster in IFA with the same dose of antigen after 3 weeks
again after 3 weeks of booster i give a final booster with the same amount of antigen
in PBS intraperitoneally and fuse the cells after 5 days.
while screening after fusion i use anti-IgG-HRP conjugate (Sigma) and TMB
substrate
in the initial screenings i see very low OD of positives but later i loose them
ofcouse i never checked for anti-IgM antibodies
may be i will do this time
is it worthy trying with rats for immunization also as suggested by one of the members before
any suggestions from anyone would be greatly appreciated
thanking you in advance
leelaram

hi,
as i mentioned in my previous post and discussion from shiva kesava,
i wud reduce the protein quantity in boosters n i will keep an eye on immunoglobulin class.
hope for the best.

u r welcome
gud luk

hi leelaram! here are few tips that might improve your fusion process
1. as mentioned, try decreasing the antigen amount,

2. while checking the titre, check for all classes of antibodies (if not all at least M,G and A) and also while screening.

3. most importantly i would like to suggest you one thing. The efficiency of final boost increases as sc < ip < iv. (iv= good ; ip= fair ; sc=poor)
you generally give final booster 3 days before fusion. its purpose is to rapidly expand the B cell population and for this antigen should be readily available for antigen presentation. iv is the most suitable route for this purpose. other cases are also used depending on some specific requirements and conditions but antigen is slowly exposed to the immune system and the response is slow and thus are less prefered routes. so try giving the final boost intravenously.

do you supplement spent media of sp2/Ocell line to your hybridoma clones? if not try using 20% spent media. this will enhance the survival of your clones.
all the best