Immunization for Monoclonals

David L. Haviland, Ph.D. HAVILAND at KIDS.WUSTL.EDU
Mon May 2 15:32:09 EST 1994


Greetings one and all:

I have a question for anyone involved with the production of monoclonals.  
I did my graduate work in a lab where monoclonals were produced and it was 
a part of my dissertation (upshot: I'm familiar with the method).  Many
moons ago, when I learned the protocol I took parts of the method as
"truth" (axioms) but have recently come to question those "truths". 

#1 To the point: How critical is it that the final boost be done i.v.?

I've heard rumors that equally good production of MAbs can be had with a 
final injection of Ag in PBS given i.p.

So I decided to give it a try:
In all honesty I must confess that I elected not to do an i.v. injection 
with the mice for my last fusion.  I elected to let an in house fusion
center do the actual fusion due to time constraints on my part.  My 
immunization protocol was as follows: 1st - 30 ug of Ag in FCA, 2nd - 30 ug 
of Ag in FIC 3 weeks later, 3rd - 30 ug of Ag in FIC 3 weeks later, 4th - a 
final i.p. injection of 30 ug of Ag in PBS 5 days before fusion.  On day 
three before fusion, serum was obtained from both mice which titered out
1:50,000 before falling off.  Significant OD's were observed out to
1:300,000.  In my book - the mice were pretty well immunized as that was
the best titer I've ever had and I initially didn't believe the titers 
myself until they were repeated.  Unfortunately, something technical went
astray in the fusion itself and percentage of growth and hybrids that are
positive against the Ag were suboptimal.  I'm lucky that I have 1 reactive
MAb at all from this fusion. 

My reason for questioning the i.v. injection is that I've had the 
experience and have talked to a few people that inadvertently given the 
mouse (mice) an embolism.  In these cases, fusions had to be done 2-3 days 
earlier than planned or not at all when the mice were sick or died.  When
the fusions were done early, the production and quality of MAbs were
equally disastrous! 

Second, I've also heard a rumor (that made sense) that fusion efficiency
of immune spleen cells increases if the spleen cells had been frozen in LN2
first.  The thought being that the DMSO makes the spleen cell membranes
sticky and facilitates the PEG fusion with the fusion partner cell. 

Any comments or discussion would be appreciated.

Many thanks in advance.

David
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