ANTIBODY-BASED TECHNIQUES - A Guide to Methods in the Biomedical Sciences - page 74

was often effective. However, while one treatment was generally toler-
ated by patients, a second injection often led to death, a reaction called
“serum sickness”. Serum sickness is a hypersensitivity reaction with
symptoms similar to anaphylaxis that results from complications caused
by circulating soluble immune complexes formed between the infused
horse antibodies and the human anti-horse antibodies which deposit
into vital organs. As a result, a search for a source for human antibodies
against defined antigens has ensued.
However, no one has successfully developed a system to produce fully
human monoclonal antibodies with any reliability. Some human antibod-
ies are produced by cells transformed by the Epstein Barr Virus (EBV,
the causative agent of mononucleosis) but these are not produced in
sufficient quantities to be particularly useful, and rarely have a desired
specificity. Consequently, mouse antibodies are often re-engineered by
recombinant DNA techniques to “humanize” the antibodies, with the goal
of retaining antigen specificity but otherwise being fully or near fully hu-
man in sequence. Therefore, sequences that encode the antigen binding
activity of the antibody are retained from the mouse sequence, but the
constant regions of the heavy and light chains and, in some cases the
“framework” regions within the variable region, those giving antibodies
their characteristic structure, are replaced with sequences common to
human antibodies. Humanized antibodies are much less immunogenic
than their mouse counterparts in humans and have longer half-lives
when injected. Some examples of humanized antibodies that have been
used in patients include Rituximab which is used in treating
B cell non-Hodgkins lymphoma, and Trastuzumab used

for treatment of metastatic breast cancer.

Phage display

Phage display is a method that incorporates the ability to genetically
manipulate bacteriophage and incorporate the gene elements encoding
the variable regions of an antibody molecule. The antibody gene seg-
ments are fused with the gene encoding the coat protein and thus the
antibodies are expressed in the phage. Phages can be produced with
a wide range of potential specificities using random sequences. Phage
with the desired specificity can then be isolated on the antigen, cloned,
and the V genes present identified for further use.

C. Purification and use of antibodies

In order to obtain large quantities of polyclonal or monoclonal anti-
bodies, it is often necessary to isolate them from serum or tissue culture