Monoclonal Antibodies: Methods and Protocols

230 Pages·2007·2.48 MB·English

by  Eugene Mechetner (auth.)

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Development and Characterization of Mouse
Hybridomas
Eugene Mechetner
Summary
Cell fusion protocols that were developed by Kohler and Milstein in the mid-1970s and
aimed at producing and characterization of mouse monoclonal antibodies (MAbs) remain
the gold standard of hybridoma development. Despite tremendous progress in using MAbs
in multiple research,diagnostic,and therapeutic areas,major experimental flaws in design-
ing and carrying out hybridoma experimentation often result in the production of hybrido-
mas exhibiting poor growth parameters and secreting low-specificity and low-affinity
antibodies. This methodology chapter is built around the conventional hybridoma protocol,
with a special emphasis on tissue culture and biochemical techniques aimed at producing
truly monospecific and highly active mouse MAbs.
Key Words:Hybridoma; monoclonal antibodies; MAbs; mouse; tissue culture.
1. Introduction
Tremendous progress in the development,characterization,and manufactur-
ing of monoclonal antibodies (MAbs) has been made since 1976,the year when
George J. F. Kohler and Cesar Milstein published their seminal paper (1)on the
production of MAbs by producing hybrids between mouse splenocytes with
their myeloma fusion partner. Kohler’s and Milstein’s outstanding contribution,
for which they were awarded together with Niels K. Jerne the 1984 Nobel Prize
in Physiology or Medicine,and—beyond all—their deliberate (and,alas,incom-
prehensible by today’s standards) decision not to patent the hybridoma technol-
ogy resulted in the rapid and widespread adoption of MAbs by both academia
and industry.
As shown in Table 1,over the last 30 yr two new types of MAbs,recombinant
and synthetic, have been developed and validated. Recombinant MAbs can be
From: Methods in Molecular Biology, vol. 378: Monoclonal Antibodies: Methods and Protocols
Edited by: M. Albitar © Humana Press Inc., Totowa, NJ
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