Biology Reference
In-Depth Information
12
Depending on the total number of sequences used in the calculation, CDR's
have higher values of variability, about 50 to 150, and FR's lower values,
less than 30 as in the case of cytochrome c's. In addition, amino acid
residues just in front and just after CDR's are usually invariant or nearly
invariant. Even though nucleotide sequences of antibody genes would not
be determined until eight years later, some basic mechanisms of antibody
production became imaginable. During the subsequent 30 years, the rapid
development of genetic and protein engineering technology opened up the
possibility of artificially synthesizing designer antibodies for therapeutic
uses.
VERIFICATION OF PREDICTIONS
Some of the immunoglobulins from mouse models of multiple myeloma
were soon isolated, purified and crystallized. However, what antigens these
immunoglobulins would bind were not known. Their three dimensional
structures, as determined by X-ray diffraction studies, showed that the six
CDR's were indeed physically located together on one surface of the
molecule. The third CDR's of both light and heavy chains were in the
middle of the site, while the second CDR's on the periphery. They could
indeed bind foreign antigen molecules.
The actual verification of our prediction would, however, wait for the
development of a new technology, namely the production of monoclonal
antibodies. Kohler and Milstein (1975) developed a method of producing
antibodies of a defined specificity in a continuous culture. Thus, when a
mouse is injected with a specific antigen, e.g. lysozyme, its spleen cells can
be fused with myeloma cells to generate such continuous cultures known as
hybridoma cell lines. The antibody produced by each cell line is uniform,
like the myeloma proteins, except that its antigen is known. Usually,
various anti-lysozyme antibodies with different binding affinities can be
isolated. In most cases, complete antibodies are difficult to crystallize due
to the flexible nature of their hinge regions. Thus, their Fab fragments are
manufactured by papain digestion (Porter, 1959), and are co-crystallized
with the antigen molecule. If the binding affinity of the Fab fragment for
lysozyme is sufficient high, they would form stable crystals. Eventually, 15
years later, with the development of the this technology, an anti-lysozyme
antibody Fab fragment was co-crystallized with lysozyme (Fig. 1-6), so that
their contacting amino acid residues could be analyzed in detail (Amit
et
al
., 1986).
