Hybridoma stability - Hybridoma (May/06/2004 )

Hi Guys
I am having real difficulty producing a monoclonal to a particular target. When I immunise the mice I get a really got antibody titre. When it comes to fusion I can't get a positive hybridoma (seven attempts at a fusion to date). My myeloma partner is SP2 which has worked well on 8 or 9 other hybridomas I have developed over the past few months. I am absolutely mistified as to why I can't get this to work.

All ideas would be much appreciated.

Mabman

Try KLH conjugation, change species ( rat to mouse or vice versa) - Titermax SQ is a lot better than Freund's in my experience too......I wouldn't blame the SP2/0 - focus on your antigen -

Also immunize 3x and wait ~ 6 months - then quick final boost and fuse - some projects I've worked on worked much better after a long sit - I think high titer can screw up the B-cell population for certain strange targets.

I have a similar problem. Immunized mice had a good antibody titre but after cell fusion I m'having a small antibody response to the target antigen. When I changed the secondary antibody specific for mouse IgG for a secondary antibody specific for mice IgG-IgM-IgA I found it positive reactivity in more wells. Mainly, I have IgM-secreting antibodies, but I don't know because I can't found IgG positive hybridomas.

tonix37

hi,
i have question that is, did u get any success with same sp2o cells after the fusion which did not worked?
if other fusion worked, then we dont doubt cell line
if u did not try other fusion then i certainly go for second cell line, for example p3x63 cell line.

in other point of view,
in my experience if we get high titre Ab in blood, i got bad results after fusion. for example most of the clones were of igM type or they used to loose Ab secretion capacity afte a while.
try giving low amount of antigen, and let animal get boosted with small amounts of protein(by using low anitigen conc you bring high specificity of antibodies). if you give, too much of antigen to mice, it may show high titres of antibodies may be those are igM isotypes at the time of testing but at the end after fusion u may not be finding any ab secretion.

i hope this info atleast helped u understand the situation, if you are interested in y igM are produced at the first place, then u can get the answer in antibody diversity process.

gud luck

hi Mabman ! let me tell you that in the preparation of monoclonals there is also chance factor involved.
1.you see B cell specific to your epitope should participate in the fusion process. more the no of B cell clones for your specific epitopes more is the chances of your B cell of interest participating in the fusion. so proper immunization is essential for a sucessful fusion.
2. screening is the next big but a very important task. most people loose their clones during screenings. as we all know class switching occurs in a sequence ( Ig M D G E A), the first type is usually IgM. as the B cell matures and class switcing occurs it produces lot of IgG Abs. i think you are loosing your clones during screening. are you using secondary antibodies for all the classes of immunoglobulins?
3. i donot find any fault with your SP2/O fusion partner.
4. try using spleen as well as lymph nodes ( sites of antigen presentation and B cell cloning) for fusion.
all the best for your next fusion.

hi everybody,

i have heard that even a little infection of animals can completely abolish the fusion...

Seb

i think post#5 give a good propose, immunization is very immportant to get McAb

hi everybody,
even i am facing the same problem like mabman
i am getting very good titres to the antigen but after fusion i am not able to get any positives
ofcourse this is the first time i am doing a mab work
so is it helpful to decrease the dose of the antigen?
i will try checking the isotype of the anitbody as suggested by others in this forum before
anyway i am doing a positive control fusion i.e., i am trying to raise monoclonal antibodies
to some other unrelated protein to check if there is any technical problem or is it because of
the antigen i am using
any suggestions would greatly help me as i am on this job since 2 years
thanx

hi,

so is it helpful to decrease the dose of the antigen?

it is also important to give appropriate doses of antigen, especially when u give boosting doses, u should use less antigen so that the B cells which are specific to ur particular antigen alone get activated and start producing antibodies.

in my experience i used to immunize minimum 3 animals and take one which give moderate levels of Ab titre rather taking the one whihc give extream titres.

gud luk

hi payeli,
thanks for your suggestion
can you tell me what is the amount of antigen you immunized per mouse both
for primary and booster doses
and what is the titre of mouse which u used for sacrificing
thanx